Marine diatoms are known to be responsible for about a quarter of global primary production and their photosynthesis is sustained by inorganic carbon-concentrating mechanisms and/or C 4 metabolism. Activities of the inorganic carbon-concentrating mechanism are attenuated under enriched [CO 2 ]; however, impacts of this factor on primary productivity and the molecular mechanisms of CO 2 responses in marine diatoms are unknown. In this study, transgenic cells were generated of the marine diatom Phaeodactylum tricornutum by the introduction of a b-glucuronidase reporter gene under the control of an intrinsic CO 2 -responsive promoter, which is the sequence between 280 to 161 relative to the transcription start site of a chloroplasticcarbonic anhydrase gene, ptca1, obtained from P. tricornutum. The activity of the ptca1 promoter was effectively repressed in air-level CO 2 by treating cells with a 1.0 mM cAMP analog, dibutyryl cAMP, or a cAMP phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine. Deletion of the intrinsic cAMP-response element from the ptca1 promoter caused a lack of repression of the reporter gene uidA, even under elevated [CO 2 ] and a null phenotype to the strong repressive effects of dibutyryl cAMP and 3-isobutyl-1-methylxanthine on the ptca1 promoter. Deletion of the cAMP-response element was also shown to cause derepression of the uidA reporter gene in the dark. These results indicate that the cytosolic cAMP level increases under elevated [CO 2 ] and represses the ptca1 promoter. This strongly suggests the participation of cAMP metabolism, presumably at the cytosolic level, in controlling CO 2 -acquisition systems under elevated [CO 2 ] at the ocean surface in a marine diatom.Marine diatoms are responsible for one-half of primary productivity in the ocean and hence play a key role in global cycles of carbon and other inorganic nutrients (Tréguer et al., 1995;Falkowski et al., 2000).[CO 2 ] dissolved in seawater is limited under the present atmospheric pCO 2 (below 15 mM at 20°C) that is much lower than the K m [CO 2 ] of Rubisco in diatom species (Badger et al., 1998). This implies that marine diatoms need active uptake and accumulation systems for dissolved inorganic carbon (DIC) to support their photosynthesis. There is a substantial body of evidence that the operation of the inorganic carbon-concentrating mechanism (CCM) confers on marine diatom cells highaffinity photosynthesis for DIC (Colman and Rotatore, 1995;Johnston and Raven, 1996;Matsuda et al., 2001), which is due to the operation of active uptake of both CO 2 and HCO 2 3 (Colman and Rotatore, 1995;Johnston and Raven, 1996;Matsuda et al., 2001). The activity of the CCM is suppressed under CO 2 -enriched conditions, whereas it is induced in CO 2 -limiting conditions; this regulation is due to CO 2 sensing by algal cells and induction of the CCM facilitates an ample supply of CO 2 to Rubisco even under extreme [CO 2 ] limitation (Badger et al., , 1998Kaplan et al., 1980;Miller et al., 1990;Colman and Rotatore, 1995;Johnston and R...

Promoter sequences of the cytomegalovirus (PCMV), the rous sarcoma virus long terminal repeat (PRSV-LTR) and the cauliflower mosaic virus 35s (PCaMV35s) were ligated with the beta-glucuronidase (GUS) gene, uidA, and were introduced into cells of the marine diatom, Phaeodactylum tricornutum. Transformants were selected on a 100 mg l(-1) Zeocin plate, and Zeocin-resistant clones were further selected by the occurrence of GUS activity. Two to 10 GUS-positive clones were obtained, and GUS activities in these transformants did not change in response to changes in ambient CO(2) concentration except that the PRSV-LTR was weakly activated in air. These results indicate that a wide spectrum of viral promoters originating from mammalian, avian and plant hosts can operate as constitutive promoters in a marine diatom. The CO(2) responsive promoter sequence of the chloroplastic carbonic anhydrase gene in P. tricornutum (Pptca1) with a deleted initiator region was ligated with the minimal region of the PCMV followed by uidA and was introduced into P. tricornutum. GUS expression in the resulting transformants was clearly regulated by CO(2), that is, GUS expression was stimulated in air to about 10-fold than that in cells grown in 5% CO(2). However, the CO(2) response disappeared when the core regulatory region of Pptca1 (-76 to -11 bp) was removed. The regulative function of the endogenous diatom promoter was thus maintained after fusion with an extrinsic viral promoter. These results indicate that diatom cells accommodate a wide range of transcriptional system from beyond the plant kingdom and that an efficient transcriptional system could potentially be constructed in marine diatoms by selecting an appropriate set of viral promoter and functional cis elements.