Regulation of antioxidant enzymes is critical to control the levels of reactive oxygen species in cell compartments highly susceptible to oxidative stress. In this work, we studied the regulation of a chloroplastic iron superoxide dismutase (Fe-SOD) from Lingulodinium polyedrum (formerly Gonyaulax polyedra) under different physiological conditions. A cDNA-encoding Fe-SOD was isolated from this dinoflagellate, showing high sequence similarity to cyanobacterial, algal, and plant FeSODs. Under standard growth conditions, on a 12:12-h light-dark cycle, Lingulodinium polyedrum Fe-SOD exhibited a daily rhythm of activity and cellular abundance with the maximum occurring during the middle of the light phase. Northern analyses showed that this rhythmicity is not related to changes in Fe-SOD mRNA levels, indicative of translational regulation. By contrast, conditions of metal-induced oxidative stress resulted in higher levels of Fe-SOD transcripts, suggesting that transcriptional control is responsible for increased protein and activity levels. Daily (circadian) and metalinduced up-regulation of Fe-SOD expression in L. polyedrum are thus mediated by different regulatory pathways, allowing biochemically distinct changes appropriate to oxidative challenges. Reactive oxygen species (ROS)1 such as superoxide (O 2 . ), and in some cases hydrogen peroxide (H 2 O 2 ), are normal by-products of oxidative metabolism and have the potential to give rise to hydroxyl radicals (HO ⅐ ). Although some ROS may function as important signaling molecules that alter gene expression and modulate the activity of specific defense proteins (1), all ROS may be harmful and pose a threat to aerobic organisms. Oxidative damage to DNA, proteins, and lipids can lead to mutagenesis, carcinogenesis, and alterations in cell structure (2). Organisms combat toxic effects of oxygen with antioxidants, which include detoxifying enzymes and low molecular weight compounds. The enzyme superoxide dismutase (SOD) represents a first step in such ROS scavenging systems. SOD isoforms, including the copper/zinc-containing (CuZn-SOD), manganese-containing (Mn-SOD), and iron-containing (Fe-SOD) metalloenzymes, catalyze the dismutation of O 2. to H 2 O 2 and oxygen. In photosynthetic eukaryotes, CuZn-SOD is usually located in the cytosol and extracellular space, although some plants also possess a chloroplastic CuZn-SOD isoform. Mn-SOD and Fe-SOD are found within the mitochondria and chloroplast, respectively (3).Irradiation by visible light in the presence of a photosensitizer leads to the production of ROS, which in plants and algae is linked to photosynthesis (4). Because of the elevated oxygen concentration and intense electron flux within chloroplasts, electrons inevitably react with oxygen, thereby generating O 2 . , which dismutates to oxygen and H 2 O 2 , producing the highly reactive HO ⅐ through the metal ion catalyzed Haber-Weiss reaction (5). Even under nonstress conditions, this ROS-generating mechanism can do harm and inactivate the photosystem II reaction ...

The circadian clock has previously been shown to restrict synthesis of several proteins in the dinoflagellate Gonyaulax polyedra to only a few hours each day. We have identified one of these proteins as glyceraldehyde-3-phosphate dehydrogenase. Two nuclear genes encoding the enzyme have been cloned, one corresponding to a cytoplasmic isoform and the other to a plastid targeted protein. On the basis of protein microsequence data, we conclude that the synthesis of the plastid isoform is clock-regulated. This regulation is not related to mRNA levels, which remain constant throughout the cycle, suggesting a translational control mechanism, in contrast to the transcriptional regulation of GAPDH that has been demonstrated in Neurospora. Although the rhythm of synthesis has a high amplitude, the abundance and activity rhythms are greatly attenuated, which is attributed to the long half-life of the protein.

The primary structure of mitrocomin consists of 190 amino acid residues, with three Ca*"-binding sites and a tyrosine residue at the C-terminus. Mitrocomin shows an amino acid sequence homology of 67.9% and 60.7% when compared with aequorin and clytin, respectively. The amino acid residues Cys '52, His", His'6g, Trpi2, Trp", TI$"~, Trl? and Tipi73 are conserved in all three photoproteins, suggesting that they play a role in light emission.

Alignment of the primary structures of the hydrozoan photoproteins, aequorin, mitrocomin, clytin and obelin showed very strong amino acid sequence identities. The Ca(2+)-binding sites of the proteins were found to be highly conserved. The Ca(2+)-binding sites were also homologous to the Ca(2+)-binding sites of other Ca(2+)-binding proteins. However, aequorin, mitrocomin, clytin and obelin differed from other Ca(2+)-binding proteins in that they contained a relatively large number of cysteine, tryptophan, histidine, proline and tyrosine residues, suggesting that these residues may have evolved as part of the light-emitting mechanism. Construction of a phylogenetic tree showed that aequorin, mitrocomin, clytin and obelin form a closely related group of proteins.