Characterization of iron-responsive promoters in the marine diatom Phaeodactylum tricornutum

It is well established that iron is one of the major constraints of primary productivity of marine diatoms in world oceans. In the present study, changes in the transcript levels of the 20 iron related genes were profiled in the marine diatom Phaeodactylum tricornutum during an early stage of acclimation from iron replete to iron-limited conditions. The results clearly showed that the profiles differ depending on genes, suggesting the occurrence of several modes of iron-responsive regulation at the transcriptional level. Upstream DNA sequences of iron starvation induced protein1 (Isi1), ferrichrome binding protein1 (FBP1), and flavodoxin (Fld) genes were isolated, fused with the GUS reporter gene, uidA, and transformed into P. tricornutum. Obtained transformants were subjected to the GUS reporter assay and the result clearly revealed that the GUS activity of all transformants was significantly increased upon iron limitation. Iron responsive Cis-elements in each promoter region were determined by the promoter truncation technique, demonstrating the occurrence of the critical iron-responsive regulatory regions of about 30bp in the promoter regions of three genes, Isi1, FBP1, and Fld. Interestingly, these sequences were similar with each other revealing two conserved motifs, A; A(A/C)G(G/C)C(G/-)C(A/G)TG; and B; CACGTG(T/C)C, which are homologous to the iron responsive Cis-element in the green alga, Chlamydomonas reinhardtii. The impairment of the motif B in the Isi1 promoter resulted in the loss of iron response and the core regulatory region of the FBP1 promoter conferred an iron response on the constitutive cytomegalovirus promoter, PCMV, indicating that these conserved promoter sequences are iron-responsive elements. Finally, the inductive regulation of these promoters under iron-limited conditions was dissipated specifically by 5% CO2, strongly suggesting the participation of CO2 in the transcriptional regulation of the iron-related gene promoters.