Localization of Soluble β-Carbonic Anhydrase in the Marine Diatom Phaeodactylum tricornutum. Sorting to the Chloroplast and Cluster Formation on the Girdle Lamellae  

Abstract

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.

Light and CO2/cAMP Signal Cross Talk on the Promoter Elements of Chloroplastic β-Carbonic Anhydrase Genes in the Marine Diatom Phaeodactylum tricornutum

“…Numerous CAs, a critical CCM component, were also found in the genomes of P. tricornutum and T. pseudonana and most of them have been localized Tanaka et al, 2005;Tachibana et al, 2011;Samukawa et al, 2014). Interestingly, there are significant differences in the location of CAs between these two species.…”

“…PtCA2 is a paralogous CA to PtCA1, with an approximately 80% amino acid sequence identity whose transcription is also CO 2 -responsive, and they colocalize at the pyrenoid of P. tricornutum Tanaka et al, 2005;Tachibana et al, 2011). Both CAs are under redox regulation by the function of diatom plastidic thioredoxins (Kikutani et al, 2012) and the function of the promoters of both CA genes are responsive to CO 2 via the function of cAMP ( Fig.…”

Identification and Preliminary Characterization of Two cDNAs Encoding Unique Carbonic Anhydrases from the Marine Alga Emiliania huxleyi

“…The use of molecular and genetic approaches to study inorganic carbon metabolism and CCMs in other unicellular freshwater and marine algae has revealed an incredible diversity of carbonic anhydrases representing all known classes of these enzymes (12,15,18,19,30,35,39,43). For example, the green alga Chlamydomonas reinhardtii possesses six CAs representing two classes (␣ and ␤) with distinct cellular locations: the periplasm (Cah1 and Cah2), thylakoid lumen (Cah3 and Cah6), and mitochondria (ca1 and ca2) (19).…”

CO2 Sensing at Ocean Surface Mediated by cAMP in a Marine Diatom

“…A b-carbonic anhydrase in the marine diatom P. tricornutum (PtCA1), thought to be one of the crucial chloroplastic components of the CCM (Satoh et al, 2001;Tanaka et al, 2005), is known to be regulated strictly by the ambient [CO 2 ] and light . A promoter region of the PtCA1 gene (ptca1) was isolated previously, and it was demonstrated that the critical CO 2 -response sequence was located downstream 270 bp relative to the transcription start site .…”