MATERIALS AND METHODSAfter cold treatment of seedlings of winter wheat (Triticum aestivum L.), levels of hydrogen peroxide in the leaves were measured. The concentration of hydrogen peroxide increased to about three times the control level within a few minutes, and retumed to the normal level in 15 to 20 minutes. The elevated level of hydrogen peroxide was found to be equivalent to 1.5 micromoles per gram fresh weight tissues of leaves.Chemicals and Enzyme DMAB and MBTH were purchased from Nakarai Tesque,

The algal pyrenoid is a large plastid body, where the majority of the CO 2 -fixing enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) resides, and it is proposed to be the hub of the algal CO 2 -concentrating mechanism (CCM) and CO 2 fixation. The thylakoid membrane is often in close proximity to or penetrates the pyrenoid itself, implying there is a functional cooperation between the pyrenoid and thylakoid. Here, GFP tagging and immunolocalization analyses revealed that a previously unidentified protein, Pt43233, is targeted to the lumen of the pyrenoid-penetrating thylakoid in the marine diatom Phaeodactylum tricornutum. The recombinant Pt43233 produced in Escherichia coli cells had both carbonic anhydrase (CA) and esterase activities. Furthermore, a Pt43233:GFP-fusion protein immunoprecipitated from P. tricornutum cells displayed a greater specific CA activity than detected for the purified recombinant protein. In an RNAi-generated Pt43233 knockdown mutant grown in atmospheric CO 2 levels, photosynthetic dissolved inorganic carbon (DIC) affinity was decreased and growth was constantly retarded; in contrast, overexpression of Pt43233:GFP yielded a slightly greater photosynthetic DIC affinity. The discovery of a θ-type CA localized to the thylakoid lumen, with an essential role in photosynthetic efficiency and growth, strongly suggests the existence of a common role for the thylakoid-luminal CA with respect to the function of diverse algal pyrenoids. marine diatom | CGHR domain | luminal carbonic anhydrase | CO 2 -concentrating mechanism | pyrenoid M arine diatoms are major primary producers, which are responsible for up to 20% of annual global carbon fixation (1, 2). To overcome the difficulties of CO 2 limitation in alkaline and high-salinity seawater, diatoms use a CO 2 -concentrating mechanism (CCM) for the intracellular accumulation of dissolved inorganic carbon (DIC) (3). It is known that the marine pennate diatom, Phaeodactylum tricornutum, uses solute carrier 4 (SLC4) family transporters to take up HCO 3 − actively from the surrounding seawater (4). Based upon physiological measurements of cellular DIC flux, it has been hypothesized that accumulated HCO 3 − is further concentrated in the chloroplast and that an ample flux of CO 2 to ribulose-1,5-bisphosphate carboxylase/ oxygenase (RubisCO) is facilitated by the pyrenoidal β-carbonic anhydrases (CAs), PtCA1 and PtCA2 (5, 6). In this process, α-type CAs present in the matrices of the four-layered chloroplast membranes are thought to prevent leakage of CO 2 from the chloroplast in P. tricornutum (7,8).Algal CCMs are distinct from their carboxysomal counterparts in cyanobacteria, and were most likely acquired by an extensive convergent evolution process (9). It is postulated that the algal CCM is composed of active DIC transport systems at the plasma membrane and the chloroplast envelope, as well as a highly localized CO 2 formation system within close proximity to RubisCO. The possibility remains that the latter process occurs within the py...

It is believed that intracellular carbonic anhydrases (CAs) are essential components of carbon concentrating mechanisms in microalgae. In this study, putative CA-encoding genes were identified in the genome sequences of the marine diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana. Subsequently, the subcellular localizations of the encoded proteins were determined. Nine and thirteen CA sequences were found in the genomes of P. tricornutum and T. pseudonana, respectively. Two of the β-CA genes in P. tricornutum corresponded to ptca1 and ptca2 identified previously. Immunostaining transmission electron microscopy of a PtCA1:YFP fusion expressed in the cells of P. tricornutum clearly showed the localization of PtCA1 within the central part of the pyrenoid structure in the chloroplast. Besides these two β-CA genes, P. tricornutum likely contains five α- and two γ-CA genes, whereas T. pseudonana has three α-, five γ-, four δ-, and one ζ-CA genes. Semi-quantitative reverse transcription PCR performed on mRNA from the two diatoms grown in changing light and CO(2) conditions revealed that levels of six putative α- and γ-CA mRNAs in P. tricornutum did not change between cells grown in air-level CO(2) and 5% CO(2). However, mRNA levels of one putative α-CA gene, CA-VII in P. tricornutum, were reduced in the dark compared to that in the light. In T. pseudonana, mRNA accumulation levels of putative α-CA (CA-1), ζ-CA (CA-3) and δ-CA (CA-7) were analyzed and all levels found to be significantly reduced when cells were grown in 0.16% CO(2). Intercellular localizations of eight putative CAs were analyzed by expressing GFP fusion in P. tricornutum and T. pseudonana. In P. tricornutum, CA-I and II localized in the periplastidial compartment, CA-III, VI, VII were found in the chloroplast endoplasmic reticulum, and CA-VIII was localized in the mitochondria. On the other hand, T. pseudonana CA-1 localized in the stroma and CA-3 was found in the periplasm. These results suggest that CAs are constitutively present in the four chloroplastic membrane systems in P. tricornutum and that CO(2) responsive CAs occur in the pyrenoid of P. tricornutum, and in the stroma and periplasm of T. pseudonana.

Photosynthesis in marine diatoms is a vital fraction of global primary production empowered by CO 2 -concentrating mechanisms. Acquisition of HCO 3− from seawater is a critical primary step of the CO 2 -concentrating mechanism, allowing marine photoautotrophic eukaryotes to overcome CO 2 limitation in alkaline high-salinity water. However, little is known about molecular mechanisms governing this process. Here, we show the importance of a plasma membrane-type HCO 3 − transporter for CO 2 acquisition in a marine diatom. Ten putative solute carrier (SLC) family HCO 3 − transporter genes were found in the genome of the marine pennate diatom Phaeodactylum tricornutum. Homologs also exist in marine centric species, Thalassiosira pseudonana, suggesting a general occurrence of SLC transporters in marine diatoms. Seven genes were found to encode putative mammalian-type SLC4 family transporters in P. tricornutum, and three of seven genes were specifically transcribed under low CO 2 conditions. One of these gene products, PtSLC4-2, was localized at the plasmalemma and significantly stimulated both dissolved inorganic carbon (DIC) uptake and photosynthesis in P. tricornutum. DIC uptake by PtSLC4-2 was efficiently inhibited by an anion-exchanger inhibitor, 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid, in a concentration-dependent manner and highly dependent on Na + ions at concentrations over 100 mM. These results show that DIC influx into marine diatoms is directly driven at the plasmalemma by a specific HCO 3 − transporter with a significant halophilic nature.bicarbonate transporter | chromista | marine environment | sodium-dependent I norganic carbon entry into algal cells is the primary limiting factor for photosynthesis and requires specific transporters (1). The problem is exacerbated especially in marine environment. Specifically, dissolved CO 2 concentrations are low, and the rate of spontaneous CO 2 formation from HCO 3 − is much slower in the ocean relative to freshwater because of the high alkalinity and salinity of seawater (2). Marine diatoms are responsible for one-fifth of global primary productivity and play a key role in global cycles of carbon and other elements (3, 4). The concentration of dissolved CO 2 in seawater under the present atmospheric pCO 2 (below 15 μM at 20°C) is much lower than the K m [CO 2 ] of ribulose-1,5-bisphosphate carboxylase/oxygenase in diatom species (5). Marine diatoms are, thus, believed to rely directly or indirectly on the use of abundant levels of seawater HCO 3 − to support their primary production. The CO 2 -concentrating mechanism (CCM) has been studied extensively in cyanobacteria, and molecular characterizations have revealed a set of CCM components that completely account for the strategy of cyanobacterial tolerance of CO 2 limitation. Freshwater β-cyanobacteria possess three plasma membrane HCO 3

Physiological properties of photosynthesis were determined in the marine diatom, Phaeodactylum tricornutum UTEX640, during acclimation from 5% CO 2 to air and related to H 2 CO 3 dissociation kinetics and equilibria in artificial seawater. The concentration of dissolved inorganic carbon at half maximum rate of photosynthesis (K 0·5 [DIC]) value in high CO 2 -grown cells was 1009 mmol m -3 but was reduced three-fold by the addition of bovine carbonic anhydrase (CA), whereas in air-grown cells K 0·5 [DIC] was 71 mmol m -3 , irrespective of the presence of CA. The maximum rate of photosynthesis (P max ) values varied between 300 and 500 mmol O 2 mg Chl -1 h -1 regardless of growth pCO 2 . Bicarbonate dehydration kinetics in artificial seawater were re-examined to evaluate the direct HCO 3 -uptake as a substrate for photosynthesis. The uncatalysed CO 2 formation rate in artificial seawater of 31·65°/ oo of salinity at pH 8·2 and 25°C was found to be 0·6 mmol m -3 min -1 at 100 mmol m -3 DIC, which is 53·5 and 7·3 times slower than the rates of photosynthesis exhibited in air-and high CO 2 -grown cells, respectively. These data indicate that even high CO 2 -grown cells of P. tricornutum can take up both CO 2 and HCO 3 -as substrates for photosynthesis and HCO 3 -use improves dramatically when the cells are grown in air. Detailed time courses were obtained of changes in affinity for DIC during the acclimation of high CO 2 -grown cells to air. The development of high-affinity photosynthesis started after a 2-5 h lag period, followed by a steady increase over the next 15 h. This acclimation time course is the slowest to be described so far. High CO 2 -grown cells were transferred to controlled DIC conditions, at which the concentrations of each DIC species could be defined, and were allowed to acclimate for more than 36 h. The

Thirteen putative carbonic anhydrase (CA) genes have been identified in the marine multipolar centric diatom, Thalassiosira pseudonana, and two of these CAs have been localized previously. The first, an alpha CA (TpαCA1), was localized in the chloroplast stroma; the second, a zeta-type CA (TpζCA1), was localized to the periplasmic space. In the present study, cloning and localization of the remaining CAs were carried out. TpγCA2, TpγCA3, TpγCA4, TpγCA5, TpδCA1, TpδCA2, TpδCA3, and TpζCA1 were responsive to CO2 availability at the transcriptional level, being significantly reduced in cells grown at 0.4 % CO2, whereas TpαCA1, TpαCA2, TpαCA3, TpγCA1, and TpδCA4 transcript levels were constitutive with respect to CO2 concentration. Full-length cDNAs for TpγCA1, TpγCA2, TpγCA3, TpγCA4, TpδCA1, and TpδCA2 were isolated and fused with the enhanced-green fluorescent gene at their 3' termini. These GFP-fusion constructs were transformed into T. pseudonana, and the resulting GFP fusion products were localized using fluorescence microscopy. The δ-type TpδCA1 was localized on the periphery of the cell, strongly suggesting localization to the periplasmic space or the frustule. The δ-type TpδCA3 and the γ-type TpγCA2 were, respectively, localized in a periplastidal compartment and the cytosol. The δ-type TpδCA2, and the γ-types TpγCA1, 3, and 4 were localized in the mitochondria. The distribution of CAs in T. pseudonana contrasts notably with that of the raphid pennate diatom P. tricornutum, with likely consequences for CCM function including modes of CO2 acquisition, regions in which DIC is accumulated, and needs for minimizing CO2 leakage from the chloroplast.

A single intracellular carbonic anhydrase (CA) was detected in air-grown and, at reduced levels, in high CO 2 -grown cells of the marine diatom Phaeodactylum tricornutum (UTEX 642). No external CA activity was detected irrespective of growth CO 2 conditions. Ethoxyzolamide (0.4 mm), a CA-specific inhibitor, severely inhibited high-affinity photosynthesis at low concentrations of dissolved inorganic carbon, whereas 2 mm acetazolamide had little effect on the affinity for dissolved inorganic carbon, suggesting that internal CA is crucial for the operation of a carbon concentrating mechanism in P. tricornutum. Internal CA was purified 36.7-fold of that of cell homogenates by ammonium sulfate precipitation, and two-step column chromatography on diethylaminoethyl-sephacel and p-aminomethylbenzene sulfone amide agarose. The purified CA was shown, by SDS-PAGE, to comprise an electrophoretically single polypeptide of 28 kD under both reduced and nonreduced conditions. The entire sequence of the cDNA of this CA was obtained by the rapid amplification of cDNA ends method and indicated that the cDNA encodes 282 amino acids. Comparison of this putative precursor sequence with the N-terminal amino acid sequence of the purified CA indicated that it included a possible signal sequence of up to 46 amino acids at the N terminus. The mature CA was found to consist of 236 amino acids and the sequence was homologous to ␤-type CAs. Even though the zinc-ligand amino acid residues were shown to be completely conserved, the amino acid residues that may constitute a CO 2 -binding site appeared to be unique among the ␤-CAs so far reported.

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.