Regulation of the expression of H43/Fea1 by multi-signals.

The composition of extracellular proteins is known to be drastically changed in the unicellular green alga Chlamydomonas reinhardtii when the cells are transferred from ambient CO(2) to elevated CO(2) conditions. We previously observed very high production of the H43/Fea1 protein under high-CO(2) (0.3-3% in air) conditions. In addition, H43/Fea1 gene expression was reported to be induced under iron-deficient and cadmium-excess conditions, but it remains unclear how gene expression is regulated by multiple signals. To elucidate the regulatory mechanism of H43/Fea1 expression, this study intended to identify a high-CO(2)-responsive cis-element in a wall-deficient strain C. reinhardtti CC-400. Cells incubated in the presence of acetate in the dark, namely heterotrophically generated high-CO(2) conditions, were used for inducing H43/Fea1 gene expression following our previous study (Hanawa et al., Plant Cell Physiol 48:299-309, 2007) in Fe-sufficient and Cd-deficient medium to prevent the generation of other signals. First, we constructed a reporter assay system using transformants constructed by introducing genes with series of 5'-deleted upstream sequences of H43/Fea1 that were fused to a coding sequence of the Ars for arylsulfatase2 reporter gene. Consequently, the high-CO(2)-responsive cis-element (HCRE) was found to be located at a -537/-370 upstream region from the transcriptional initiation site of H43/Fea1. However, it still remains possible that a -724/-537 upstream region may also have a significant role in activating gene expression regulated by high-CO(2). Remarkably, a -925/-370 upstream region could successfully activate the Ars reporter gene under heterotrophically generated high-CO(2) conditions even when the sequence containing two Fe-deficiency-responsive elements was completely deleted. These results clearly showed that H43/Fea1 expression is regulated by high-CO(2) signal independently via the HCRE that is located distantly from Fe-deficient-signal responsive element, indicating that H43/Fea1 is a multi-signal-regulated gene.