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Background  The green picoalga  Ostreococcus tauri  (Prasinophyceae), which has been described as the smallest free-living eukaryotic organism, has minimal cellular ultra-structure and a very small genome. In recent years,  O. tauri  has emerged as a novel model organism for systems biology approaches that combine functional genomics and mathematical modeling, with a strong emphasis on light regulated processes and circadian clock. These approaches were made possible through the implementation of a minimal molecular toolbox for gene functional analysis including overexpression and knockdown strategies. We have previously shown that the promoter of the  High Affinity Phosphate Transporter  ( HAPT ) gene drives the expression of a luciferase reporter at high and constitutive levels under constant light.    Methodology/Principal Findings  Here we report, using a luciferase reporter construct, that the  HAPT  promoter can be finely and reversibly tuned by modulating the level and nature of phosphate in culture medium. This  HAPT  regulation was additionally used to analyze the circadian clock gene  Time of Cab expression 1  ( TOC1 ). The phenotype of a TOC1ox/CCA1:Luc line was reverted from arrhythmic to rhythmic simply by adding phosphate to the culture medium. Furthermore, since the time of phosphate injection had no effect on the phase of CCA1:Luc expression, this study suggests further that  TOC1  is a central clock gene in  Ostreococcus .    Conclusions/Perspectives  We have developed a phosphate-regulated expression system that allows fine gene function analysis in  Ostreococcus . Recently, there has been a growing interest in microalgae as cell factories. This non-toxic phosphate-regulated system may prove useful in tuning protein expression levels quantitatively and temporally for biotechnological applications.

A Phosphate-Regulated Promoter for Fine-Tuned and Reversible Overexpression in Ostreococcus: Application to Circadian Clock Functional Analysis