A b-carbonic anhydrase (CA) in the marine diatom Phaeodactylum tricornutum (PtCA1) is encoded by the nuclear genome. This enzyme was previously found to be important for the operation of photosynthesis with a high affinity for dissolved inorganic carbon. A cDNA sequence that encodes PtCA1 (ptca1) was shown to possess a presequence of 138 bp (pre138), which encodes an N-terminal sequence of 46 amino acids (Pre46AA) that does not exist in the mature PtCA1. In this study, pre138 was ligated with the enhanced green fluorescent protein (GFP) gene (egfp), and introduced into P. tricornutum by microprojectile bombardment. Subsequently, the expressed Pre46AA-GFP fusion was shown to be localized in the chloroplast stroma, whereas the expressed GFP without Pre46AA was localized in the cytoplasm. Insertion of the DNA sequence, encoding a mature region of ptca1 (mptca1) between pre138 and egfp, resulted in the formation of particles with concentrated GFP fluorescence in the stroma of P. tricornutum. These particles, 0.3 to 3.0 mm in size, were shown to be distinct from the mitochondria and localized on the surface of the putative girdle lamella. The attachment of the initial one-half of the pre138 to the mptca1-egfp fusion caused the expressed GFP fusion to accumulate in areas surrounding the chloroplast, presumably due to the presence of the endoplasmic reticulum signal encoded by the initial half-sequence and to the absence of the chloroplast transit sequence. These results indicate that PtCA1 is targeted to the stroma by the bipartite sequences of Pre46AA and that the observed GFP particles are formed specifically in the stroma due to the function of the mptca1.

Cells of the marine diatom Phaeodactylum tricornutum Bohlin (UTEX 642) grown in 5% CO 2 were transferred to air-level CO 2 in the light or dark and allowed to acclimate to air. No accumulation of the transcript of the P. tricornutum b-carbonic anhydrase 1 (ptca1) was detected in 5% CO 2 -grown cells, but ptca1 mRNA accumulated and reached a peak after 6 h acclimation to air but decreased over the next 18 h. A similar accumulation time course was observed in cells air-acclimated in the dark, except that levels of mRNA were ,50% those in the light. These results suggest that air-level [CO 2 ] is required to trigger the transcription of ptca1 and that light affects the extent of acclimation. During acclimation to air for 120 h in the light, levels of ptca1 mRNA exhibited a periodic oscillation with a cycle of about 24 h, which, however, was not reflected in protein accumulation levels. A 5#-upstream region from the transcription-start site toward 21,292 bp of ptca1 was cloned by inverse polymerase chain reaction, and 5#-truncations were carried out on this fragment. The truncated promoter regions were fused with the b-glucuronidase gene (uidA) and introduced into P. tricornutum. The promoter fragments, truncated at positions 21,292, 2824, 2484, 2225, and 270 bp, conferred on transformants clear CO 2 -responsive b-glucuronidase expressions. In contrast, the CO 2 -responsive regulation was severely impaired or completely abolished by truncations, respectively, at position 250 or 230 bp. These results indicate that critical cis-elements required for CO 2 -responsive transcription of ptca1 may be located between 270 and 230 bp relative to the transcription start site.