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Chlamydomonas reinhardtii fox1  gene encodes a ferroxidase that is involved in cellular Fe uptake and highly induced during Fe deficient conditions. In an effort to identify  fox1  promoter regulatory elements, an insertional library was generated in a transgenic  Chlamydomonas  strain (2A38) harboring an arylsulfatase (ARS) reporter gene driven by the  fox1  promoter. Mutants with a defective response to low iron conditions were selected for further study. Among these, a strain containing a disrupted  femu2  gene was identified. Activation of the  fox1  promoter by the  femu2  gene product was confirmed by silencing the  femu2  gene using RNA interference. In three  femu2  RNAi transgenic lines (IR3, IR6, and IR7), ARS reporter gene activities declined by 84.3%, 86.4%, and 88.8%, respectively under Fe deficient conditions. Furthermore,  RT -PCR analysis of both the  femu2  mutant and the RNAi transgenic lines showed significantly decreased transcript abundance of the endogenous  fox1  gene under Fe deficient conditions. Amino acid sequence analysis of the  femu2  gene product identified three potential C2H2 zinc finger (ZF) motifs and a nuclear localization study suggests that FEMU2 is localized to the nucleus. In addition, a potential FEMU2 binding site ((G/T)TTGG(G/T)(G/T)T) was identified using PCR-mediated random binding site selection. Taken together, this evidence suggests that FEMU2 is involved in up-regulation of the  fox1  gene in Fe deficient cells.

A C2H2 Zinc Finger Protein FEMU2 Is Required for fox1 Expression in Chlamydomonas reinhardtii