Photosynthesis Research (v.109, #1-3)

Carboxysomes: cyanobacterial RubisCO comes in small packages by George S. Espie; Matthew S. Kimber (7-20).
Cyanobacteria (as well as many chemoautotrophs) actively pump inorganic carbon (in the form of HCO3 ) into the cytosol in order to enhance the overall efficiency of carbon fixation. The success of this approach is dependent upon the presence of carboxysomes—large, polyhedral, cytosolic bodies which sequester ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) and carbonic anhydrase. Carboxysomes seem to function by allowing ready passage of HCO3 into the body, but hindering the escape of evolved CO2, promoting the accumulation of CO2 in the vicinity of RubisCO and, consequently, efficient carbon fixation. This selectivity is mediated by a thin shell of protein, which envelops the carboxysome’s enzymatic core and uses narrow pores to control the passage of small molecules. In this review, we summarize recent advances in understanding the organization and functioning of these intriguing, and ecologically very important molecular machines.
Keywords: Carboxysome; Cyanobacteria; Carbon concentrating mechanism; Carbonic anhydrase; RubisCO

Comparative analysis of carboxysome shell proteins by James N. Kinney; Seth D. Axen; Cheryl A. Kerfeld (21-32).
Carboxysomes are metabolic modules for CO2 fixation that are found in all cyanobacteria and some chemoautotrophic bacteria. They comprise a semi-permeable proteinaceous shell that encapsulates ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and carbonic anhydrase. Structural studies are revealing the integral role of the shell protein paralogs to carboxysome form and function. The shell proteins are composed of two domain classes: those with the bacterial microcompartment (BMC; Pfam00936) domain, which oligomerize to form (pseudo)hexamers, and those with the CcmL/EutN (Pfam03319) domain which form pentamers in carboxysomes. These two shell protein types are proposed to be the basis for the carboxysome’s icosahedral geometry. The shell proteins are also thought to allow the flux of metabolites across the shell through the presence of the small pore formed by their hexameric/pentameric symmetry axes. In this review, we describe bioinformatic and structural analyses that highlight the important primary, tertiary, and quaternary structural features of these conserved shell subunits. In the future, further understanding of these molecular building blocks may provide the basis for enhancing CO2 fixation in other organisms or creating novel biological nanostructures.
Keywords: Carboxysome; CO2 fixation; Bacterial microcompartment; Dark reactions; Cyanobacteria; Calvin cycle; Chemoautotroph

Carboxysomes, containing the cell’s complement of RuBisCO surrounded by a specialized protein shell, are a central component of the cyanobacterial CO2-concentrating mechanism. The ratio of two forms of the β-carboxysomal protein CcmM (M58 and M35) may affect the carboxysomal carbonic anhydrase (CcaA) content. We have over-expressed both M35 and M58 in the β-cyanobacterium Synechococcus PCC7942. Over-expression of M58 resulted in a marked increase in the amount of this protein in carboxysomes at the expense of M35, with a concomitant increase in the observed CcaA content of carboxysomes. Conversely, M35 over-expression diminished M58 content of carboxysomes and led to a decrease in CcaA content. Carboxysomes of air-grown wild-type cells contained slightly elevated CcaA and M58 content and slightly lower M35 content compared to their 2% CO2-grown counterparts. Over a range of CcmM expression levels, there was a strong correlation between M58 and CcaA content, indicating a constant carboxysomal M58:CcaA stoichiometry. These results also confirm a role for M58 in the recruitment of CcaA into the carboxysome and suggest a tight regulation of M35 and M58 translation is required to produce carboxysomes with an appropriate CA content. Analysis of carboxysomal protein ratios, resulting from the afore-mentioned over-expression studies, revealed that β-carboxysomal protein stoichiometries are relatively flexible. Determination of absolute protein quantities supports the hypothesis that M35 is distributed throughout the β-carboxysome. A modified β-carboxysome packing model is presented.
Keywords: Carboxysomes; RuBisCO; CCM; Carbonic anhydrase

Cyanobacteria possess an environmental adaptation known as a CO2 concentrating mechanism (CCM) that evolved to improve photosynthetic performance, particularly under CO2-limiting conditions. The CCM functions to actively transport dissolved inorganic carbon species (Ci; HCO3 and CO2) resulting in accumulation of a pool of HCO3 within the cell that is then utilised to provide an elevated CO2 concentration around the primary CO2 fixing enzyme, ribulose bisphosphate carboxylase-oxygenase (Rubisco). Rubisco is encapsulated in unique micro-compartments known as carboxysomes and also provides the location for elevated CO2 levels in the cell. Five distinct transport systems for active Ci uptake are known, including two types of Na+-dependent HCO3 transporters (BicA and SbtA), one traffic ATPase (BCT1) for HCO3 uptake and two CO2 uptake systems based on modified NADPH dehydrogenase complexes (NDH-I3 and NDH-I4). The genes for a number of these transporters are genetically induced under Ci limitation via transcriptional regulatory processes. The in-membrane topology structures of the BicA and SbtA HCO3 transporters are now known and this may aid in determining processes related to transporter activation during dark to light transitions or under severe Ci limitation.
Keywords: Cyanobacteria; CO2 concentrating mechanism; Carboxysomes; CO2-responsive genes; Photosynthesis; Bicarbonate transporters; CO2 uptake systems; Genetic regulation

The CO2-concentrating mechanism of Synechococcus WH5701 is composed of native and horizontally-acquired components by Benjamin D. Rae; Britta Förster; Murray R. Badger; G. Dean Price (59-72).
The cyanobacterial CO2-concentrating mechanism (CCM) is an effective adaptation that increases the carbon dioxide (CO2) concentration around the primary photosynthetic enzyme Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (RuBisCO). α-Cyanobacteria (those containing Form1-A RuBisCO within cso-type α-carboxysomes) have a limited CCM composed of a small number of Ci-transporters whereas β-cyanobacteria (those species containing Form-1B RuBisCO within ccm-type β-carboxysomes) exhibit a more diverse CCM with a greater variety in Ci-transporter complement and regulation. In the coastal species Synechococcus sp. WH5701 (α-cyanobacteria), the minimal α-cyanobacterial CCM has been supplemented with β-cyanobacterial Ci transporters through the process of horizontal gene transfer (HGT). These transporters are transcriptionally regulated in response to external Ci-depletion however this change in transcript abundance is not correlated with a physiological induction. WH5701 exhibits identical physiological responses grown at 4% CO2 (K 1/2 ≈ 31 μM Ci) and after induction with 0.04% CO2 (K 1/2 ≈ 29 μM Ci). Insensitivity to external Ci concentration is an unusual characteristic of the WH5701 CCM which is a result of evolution by HGT. Our bioinformatic and physiological data support the hypothesis that WH5701 represents a clade of α-cyanobacterial species in transition from the marine/oligotrophic environment to a coastal/freshwater environment.
Keywords: Horizontal gene transfer; Synechococcus ; WH5701; CO2 concentrating mechanism; Cyanobacteria

Interactions between CCM and N2 fixation in Trichodesmium by Sven A. Kranz; Meri Eichner; Björn Rost (73-84).
In view of the current increase in atmospheric pCO2 and concomitant changes in the marine environment, it is crucial to assess, understand, and predict future responses of ecologically relevant phytoplankton species. The diazotrophic cyanobacterium Trichodesmium erythraeum was found to respond strongly to elevated pCO2 by increasing growth, production rates, and N2 fixation. The magnitude of these CO2 effects exceeds those previously seen in other phytoplankton, raising the question about the underlying mechanisms. Here, we review recent publications on metabolic pathways of Trichodesmium from a gene transcription level to the protein activities and energy fluxes. Diurnal patterns of nitrogenase activity change markedly with CO2 availability, causing higher diel N2 fixation rates under elevated pCO2. The observed responses to elevated pCO2 could not be attributed to enhanced energy generation via gross photosynthesis, although there are indications for CO2-dependent changes in ATP/NADPH + H+ production. The CO2 concentrating mechanism (CCM) in Trichodesmium is primarily based on HCO3 uptake. Although only little CO2 uptake was detected, the NDH complex seems to play a crucial role in internal cycling of inorganic carbon, especially under elevated pCO2. Affinities for inorganic carbon change over the day, closely following the pattern in N2 fixation, and generally decrease with increasing pCO2. This down-regulation of CCM activity and the simultaneously enhanced N2 fixation point to a shift in energy allocation from carbon acquisition to N2 fixation under elevated pCO2 levels. A strong light modulation of CO2 effects further corroborates the role of energy fluxes as a key to understand the responses of Trichodesmium.
Keywords: CO2 concentrating mechanism; Diazotroph; Energy allocation; N acquisition; Ocean acidification; Photosynthesis

Physiological characterization and light response of the CO2-concentrating mechanism in the filamentous cyanobacterium Leptolyngbya sp. CPCC 696 by Elvin D. de Araujo; Jason Patel; Charlotte de Araujo; Susan P. Rogers; Steven M. Short; Douglas A. Campbell; George S. Espie (85-101).
We studied the interactions of the CO2-concentrating mechanism and variable light in the filamentous cyanobacterium Leptolyngbya sp. CPCC 696 acclimated to low light (15 μmol m−2 s−1 PPFD) and low inorganic carbon (50 μM Ci). Mass spectrometric and polarographic analysis revealed that mediated CO2 uptake along with both active Na+-independent and Na+-dependent HCO3 transport, likely through Na+/HCO3 symport, were employed to concentrate Ci internally. Combined transport of CO2 and HCO3 required about 30 kJ mol−1 of energy from photosynthetic electron transport to support an intracellular Ci accumulation 550-fold greater than the external Ci. Initially, Leptolyngbya rapidly induced oxygen evolution and Ci transport to reach 40–50% of maximum values by 50 μmol m−2 s−1 PPFD. Thereafter, photosynthesis and Ci transport increased gradually to saturation around 1,800 μmol m−2 s−1 PPFD. Leptolyngbya showed a low intrinsic susceptibility to photoinhibition of oxygen evolution up to PPFD of 3,000 μmol m−2 s−1. Intracellular Ci accumulation showed a lag under low light but then peaked at about 500 μmol photons m−2 s−1 and remained high thereafter. Ci influx was accompanied by a simultaneous, light-dependent, outward flux of CO2 and by internal CO2/HCO3 cycling. The high-affinity and high-capacity CCM of Leptolyngbya responded dynamically to fluctuating PPFD and used excitation energy in excess of the needs of CO2 fixation by increasing Ci transport, accumulation and Ci cycling. This capacity may allow Leptolyngbya to tolerate periodic exposure to excess high light by consuming electron equivalents and keeping PSII open.
Keywords: Ci pump/leak cycle; CO2-concentrating mechanism; CO2 uptake; Cyanobacteria; Leptolyngbya ; Na+-dependent HCO3 transport; Light response; Photoprotection; Photosynthesis

The photorespiratory pathway was shown to be essential for organisms performing oxygenic photosynthesis, cyanobacteria, algae, and plants, in the present day O2-containing atmosphere. The identification of a plant-like 2-phosphoglycolate cycle in cyanobacteria indicated that not only genes of oxygenic photosynthesis but also genes encoding photorespiratory enzymes were endosymbiotically conveyed from ancient cyanobacteria to eukaryotic oxygenic phototrophs. Here, we investigated the origin of the photorespiratory pathway in photosynthetic eukaryotes by phylogenetic analysis. We found that a mixture of photorespiratory enzymes of either cyanobacterial or α-proteobacterial origin is present in algae and higher plants. Three enzymes in eukaryotic phototrophs clustered closely with cyanobacterial homologs: glycolate oxidase, glycerate kinase, and hydroxypyruvate reductase. On the other hand, the mitochondrial enzymes of the photorespiratory cycle in algae and plants, glycine decarboxylase subunits and serine hydroxymethyltransferase, evolved from proteobacteria. Other than most genes for proteins of the photosynthetic machinery, nearly all enzymes involved in the 2-phosphogylcolate metabolism coexist in the genomes of cyanobacteria and heterotrophic bacteria.
Keywords: Cyanobacteria; Eukaryotic algae; Evolution; Photorespiration; Phylogeny; Plant

Many microalgae are capable of acclimating to CO2 limited environments by operating a CO2 concentrating mechanism (CCM), which is driven by various energy-coupled inorganic carbon (Ci; CO2 and HCO3 ) uptake systems. Chlamydomonas reinhardtii (hereafter, Chlamydomonas), a versatile genetic model organism, has been used for several decades to exemplify the active Ci transport in eukaryotic algae, but only recently have many molecular details behind these Ci uptake systems emerged. Recent advances in genetic and molecular approaches, combined with the genome sequencing of Chlamydomonas and several other eukaryotic algae have unraveled some unique characteristics associated with the Ci uptake mechanism and the Ci-recapture system in eukaryotic microalgae. Several good candidate genes for Ci transporters in Chlamydomonas have been identified, and a few specific gene products have been linked with the Ci uptake systems associated with the different acclimation states. This review will focus on the latest studies on characterization of functional components involved in the Ci uptake and the Ci-recapture in Chlamydomonas.
Keywords: CO2 concentrating mechanism; Active inorganic carbon uptake; Transporter; Bicarbonate; CO2 ; Chlamydomonas

Chlamydomonas reinhardtii and other microalgae show adaptive changes to limiting CO2 conditions by induction of CO2-concentrating mechanisms. The limiting-CO2-inducible gene, LCIB, encodes a soluble plastid protein and is proposed to play a role in trapping CO2 released by CAH3 (thylakoid lumen carbonic anhydrase) catalyzed dehydration of accumulated Ci, especially in low CO2 (L-CO2; ~0.04% CO2) conditions. To gain further insight into the mechanisms of Ci uptake and accumulation in L-CO2 acclimated C. reinhardtii, we performed an insertional mutagenesis screen to isolate extragenic suppressors that restore the growth of lcib mutants (pmp1 and ad1) in L-CO2. Four independent suppressors are described here and classified by their photosynthetic affinities for Ci and expression patterns of known limiting-CO2-inducible transcripts. Genetic analysis of the four suppressors identified two allelic, dominant suppressors (su4 and su5), and two recessive suppressors (su1 and su8). Consistent with the suppression phenotype, both the relative affinities of photosynthetic O2 evolution and internal Ci accumulation in all four suppressors were substantially increased relative to pmp1/ad1 in L-CO2 acclimated cells. The relative affinities of pmp-su1 and ad-su8 for Ci were nearly the same as wild type, but that of pmp-su4/su5 was intermediate between pmp-su1 and pmp1. Also, the interactions between lcib mutations and each of the three suppressors varied over the range of CO2 acclimation states. Our results suggest complex contributions of LCIB-dependent and independent active Ci uptake/accumulation systems in various CO2 acclimation states and therefore provide new clues about the roles played by LCIB in limiting Ci acclimation.
Keywords: CO2-concentrating mechanism; Chlamydomonas reinhardtii ; LCIB ; Ci uptake/accumulation; Bicarbonate; Inorganic carbon

The carbonic anhydrase isoforms of Chlamydomonas reinhardtii: intracellular location, expression, and physiological roles by James V. Moroney; Yunbing Ma; Wesley D. Frey; Katelyn A. Fusilier; Trang T. Pham; Tiffany A. Simms; Robert J. DiMario; Jing Yang; Bratati Mukherjee (133-149).
Aquatic photosynthetic organisms, such as the green alga Chlamydomonas reinhardtii, respond to low CO2 conditions by inducing a CO2 concentrating mechanism (CCM). Carbonic anhydrases (CAs) are important components of the CCM. CAs are zinc-containing metalloenzymes that catalyze the reversible interconversion of CO2 and HCO3 . In C. reinhardtii, there are at least 12 genes that encode CA isoforms, including three alpha, six beta, and three gamma or gamma-like CAs. The expression of the three alpha and six beta genes has been measured from cells grown on elevated CO2 (having no active CCM) versus cells growing on low levels of CO2 (with an active CCM) using northern blots, differential hybridization to DNA chips and quantitative RT-PCR. Recent RNA-seq profiles add to our knowledge of the expression of all of the CA genes. In addition, protein content for some of the CA isoforms was estimated using antibodies corresponding to the specific CA isoforms: CAH1/2, CAH3, CAH4/5, CAH6, and CAH7. The intracellular location of each of the CA isoforms was elucidated using immunolocalization and cell fractionation techniques. Combining these results with previous studies using CA mutant strains, we will discuss possible physiological roles of the CA isoforms concentrating on how these CAs might contribute to the acquisition and retention of CO2 in C. reinhardtii.
Keywords: Carbonic anhydrase; CO2 concentrating mechanism; CCM; Chlamydomonas; Pyrenoid

When CO2 supply is limited, aquatic photosynthetic organisms induce a CO2-concentrating mechanism (CCM) and acclimate to the CO2-limiting environment. Although the CCM is well studied in unicellular green algae such as Chlamydomonas reinhardtii, physiological aspects of the CCM and its associated genes in multicellular algae are poorly understood. In this study, by measuring photosynthetic affinity for CO2, we present physiological data in support of a CCM in a multicellular green alga, Volvox carteri. The low-CO2-grown Volvox cells showed much higher affinity for inorganic carbon compared with high-CO2-grown cells. Addition of ethoxyzolamide, a membrane-permeable carbonic anhydrase inhibitor, to the culture remarkably reduced the photosynthetic affinity of low-CO2 grown Volvox cells, indicating that an intracellular carbonic anhydrase contributed to the Volvox CCM. We also isolated a gene encoding a protein orthologous to CCM1/CIA5, a master regulator of the CCM in Chlamydomonas, from Volvox carteri. Volvox CCM1 encoded a protein with 701 amino acid residues showing 51.1% sequence identity with Chlamydomonas CCM1. Comparison of Volvox and Chlamydomonas CCM1 revealed a highly conserved N-terminal region containing zinc-binding amino acid residues, putative nuclear localization and export signals, and a C-terminal region containing a putative LXXLL protein–protein interaction motif. Based on these results, we discuss the physiological and genetic aspects of the CCM in Chlamydomonas and Volvox.
Keywords: CO2-concentrating mechanism; Carbonic anhydrase; CCM1; Chlamydomonas reinhardtii ; Volvox carteri

Acclimation of Chlamydomonas reinhardtii (hereafter, Chlamydomonas) to low or limiting CO2 or inorganic carbon (Ci) has been studied fairly extensively with regard to the mechanisms underlying the inducible Ci acquisition systems and the signal transduction pathway involved in recognizing and responding to decreased Ci availability. Investigation of low Ci acclimation responses typically is performed with non-synchronous cultures grown in continuous light to avoid any effects of the cell division cycle (CDC) confounding interpretation of acclimation responses. However, little is known about whether acclimation to low Ci might affect the distribution of cells among the various stages of the CDC. To investigate the effects of a limiting-Ci challenge on the CDC of Chlamydomonas, flow cytometry was used to monitor the distribution of cells among the CDC stages in both synchronous and non-synchronous cultures during acclimation to low or limiting Ci. When faced with Ci limitation, non-synchronous cultures of Chlamydomonas undergo transient synchronization as those cells past the Commitment point of the CDC undergo division, while the remainder of the cells pause their growth in early G-phase, with the result that the cells all accumulate in early G-phase, appearing transiently synchronized until acclimated sufficiently to the decreased Ci for growth to resume. This perturbation of the CDC by a limiting-Ci challenge has important implications for the interpretation of gene expression and other responses apparently induced by low or limiting Ci.
Keywords: CO2-concentrating mechanism; CCM; Chlamydomonas reinhardtii ; CAH1 ; Bicarbonate; Inorganic carbon; CDC

Regulation of the expression of H43/Fea1 by multi-signals by Masato Baba; Yutaka Hanawa; Iwane Suzuki; Yoshihiro Shiraiwa (169-177).
The composition of extracellular proteins is known to be drastically changed in the unicellular green alga Chlamydomonas reinhardtii when the cells are transferred from ambient CO2 to elevated CO2 conditions. We previously observed very high production of the H43/Fea1 protein under high-CO2 (0.3–3% in air) conditions. In addition, H43/Fea1 gene expression was reported to be induced under iron-deficient and cadmium-excess conditions, but it remains unclear how gene expression is regulated by multiple signals. To elucidate the regulatory mechanism of H43/Fea1 expression, this study intended to identify a high-CO2-responsive cis-element in a wall-deficient strain C. reinhardtti CC-400. Cells incubated in the presence of acetate in the dark, namely heterotrophically generated high-CO2 conditions, were used for inducing H43/Fea1 gene expression following our previous study (Hanawa et al., Plant Cell Physiol 48:299–309, 2007) in Fe-sufficient and Cd-deficient medium to prevent the generation of other signals. First, we constructed a reporter assay system using transformants constructed by introducing genes with series of 5′-deleted upstream sequences of H43/Fea1 that were fused to a coding sequence of the Ars for arylsulfatase2 reporter gene. Consequently, the high-CO2-responsive cis-element (HCRE) was found to be located at a −537/−370 upstream region from the transcriptional initiation site of H43/Fea1. However, it still remains possible that a −724/−537 upstream region may also have a significant role in activating gene expression regulated by high-CO2. Remarkably, a −925/−370 upstream region could successfully activate the Ars reporter gene under heterotrophically generated high-CO2 conditions even when the sequence containing two Fe-deficiency-responsive elements was completely deleted. These results clearly showed that H43/Fea1 expression is regulated by high-CO2 signal independently via the HCRE that is located distantly from Fe-deficient-signal responsive element, indicating that H43/Fea1 is a multi-signal-regulated gene.
Keywords: Chlamydomonas reinhardtii ; cis-Element; Gene expression; H43/Fea1 ; High-CO2 response; Periplasmic protein

The CO2 acquisition was analyzed in Chlamydomonas acidophila at pH 2.4 in a range of medium P and Fe concentrations and at high and low CO2 condition. The inorganic carbon concentrating factor (CCF) was related to cellular P quota (Q p), maximum CO2-uptake rate by photosynthesis ( $$ V_{{{ max },{ ext{O}}_{ 2} }} $$ ), half saturation constant for CO2 uptake (K 0.5), and medium Fe concentration. There was no effect of the medium Fe concentration on the CCF. The CCF increased with increasing Q p in both high and low CO2 grown algae, but maximum Q p was 6-fold higher in the low CO2 cells. In high CO2 conditions, the CCF was low, ranging between 0.8 and 3.5. High CCF values up to 9.1 were only observed in CO2-limited cells, but P- and CO2-colimited cells had a low CCF. High CCF did not relate with a low K 0.5 as all CO2-limited cells had a low K 0.5 (<4 μM CO2). High Ci-pools in cells with high Q p suggested the presence of an active CO2-uptake mechanism. The CCF also increased with increasing $$ V_{{{ max },{ ext{O}}_{ 2} }} $$ which reflect an adaptation to the nutrient in highest demand (CO2) under balanced growth conditions. It is proposed that the size of the CCF in C. acidophila is more strongly related to porter density for CO2 uptake (reflected in $$ V_{{{ max },{ ext{O}}_{ 2} }} $$ ) and less- to high-affinity CO2 uptake (low K 0.5) at balanced growth. In addition, high CCF can only be realized with high Q p.
Keywords: C3 photosynthesis; Micro-algae; Carbon concentrating mechanism; Phosphorus limitation; Iron toxicity

Marine diatoms, the major primary producer in ocean environment, are known to take up both CO2 and HCO3 in seawater and efficiently concentrate them intracellularly, which enable diatom cells to perform high-affinity photosynthesis under limiting CO2. However, mechanisms so far proposed for the inorganic carbon acquisition in marine diatoms are significantly diverse despite that physiological studies on this aspect have been done with only limited number of species. There are two major hypotheses about this; that is, they take up and concentrate both CO2 and HCO3 as inorganic forms, and efficiently supply CO2 to Rubisco by an aid of carbonic anhydrases (biophysical CO2-concentrating mechanism: CCM); and as the other hypothesis, biochemical conversion of HCO3 into C4 compounds may play a major role to supply concentrated CO2 to Rubisco. At moment however, physiological evidence for these hypotheses were not related well to molecular level evidence. In this study, recent progresses in molecular studies on diatom-carbon-metabolism genes were related to the physiological aspects of carbon acquisition. Furthermore, we discussed the mechanisms regulating CO2 acquisition systems in response to changes in pCO2. Recent findings about the participation of cAMP in the signaling pathway of CO2 concentration strongly suggested the occurrences of mammalian-type-signaling pathways in diatoms to respond to changes in pCO2. In fact, there were considerable numbers of putative adenylyl cyclases, which may take part in the processes of CO2 signal capturing.
Keywords: Marine diatoms; CO2 ; HCO3 transport; Carbonic anhydrase; Localization; cAMP

Localization of putative carbonic anhydrases in two marine diatoms, Phaeodactylum tricornutum and Thalassiosira pseudonana by Masaaki Tachibana; Andrew E. Allen; Sae Kikutani; Yuri Endo; Chris Bowler; Yusuke Matsuda (205-221).
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 CO2 conditions revealed that levels of six putative α- and γ-CA mRNAs in P. tricornutum did not change between cells grown in air-level CO2 and 5% CO2. 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% CO2. 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 CO2 responsive CAs occur in the pyrenoid of P. tricornutum, and in the stroma and periplasm of T. pseudonana.
Keywords: Marine diatom; Carbonic anhydrase; Inorganic carbon concentrating mechanism; Pyrenoid

High-throughput pyrosequencing of the chloroplast genome of a highly neutral-lipid-producing marine pennate diatom, Fistulifera sp. strain JPCC DA0580 by Tsuyoshi Tanaka; Yorikane Fukuda; Tomoko Yoshino; Yoshiaki Maeda; Masaki Muto; Mitsufumi Matsumoto; Shigeki Mayama; Tadashi Matsunaga (223-229).
The chloroplast genome of the highly neutral-lipid-producing marine pennate diatom Fistulifera sp. strain JPCC DA0580 was fully sequenced using high-throughput pyrosequencing. The general features and gene content were compared with three other complete diatom chloroplast genomes. The chloroplast genome is 134,918 bp with an inverted repeat of 13,330 bp and is slightly larger than the other diatom chloroplast genomes due to several low gene-density regions lacking similarity to the other diatom chloroplast genomes. Protein-coding genes were nearly identical to those from Phaeodactylum tricornutum. On the other hand, we found unique sequence variations in genes of photosystem II which differ from the consensus in other diatom chloroplasts. Furthermore, five functional unknown ORFs and a putative serine recombinase gene, serC2, are located in the low gene-density regions. SerC2 was also identified in the plasmids of another pennate diatom, Cylindrotheca fusiformis, and in the plastid genome of the diatom endosymbiont of Kryptoperidinium foliaceum. Exogenous plasmids might have been incorporated into the chloroplast genome of Fistulifera sp. by lateral gene transfer. Chloroplast genome sequencing analysis of this novel diatom provides many important insights into diatom evolution.
Keywords: Diatom; Chloroplast genome; Next-generation DNA sequencing; Lateral gene transfer; Endosymbiosis

Integration of microalgae cultivation with industrial waste remediation for biofuel and bioenergy production: opportunities and limitations by Patrick J. McGinn; Kathryn E. Dickinson; Shabana Bhatti; Jean-Claude Frigon; Serge R. Guiot; Stephen J. B. O’Leary (231-247).
There is currently a renewed interest in developing microalgae as a source of renewable energy and fuel. Microalgae hold great potential as a source of biomass for the production of energy and fungible liquid transportation fuels. However, the technologies required for large-scale cultivation, processing, and conversion of microalgal biomass to energy products are underdeveloped. Microalgae offer several advantages over traditional ‘first-generation’ biofuels crops like corn: these include superior biomass productivity, the ability to grow on poor-quality land unsuitable for agriculture, and the potential for sustainable growth by extracting macro- and micronutrients from wastewater and industrial flue-stack emissions. Integrating microalgal cultivation with municipal wastewater treatment and industrial CO2 emissions from coal-fired power plants is a potential strategy to produce large quantities of biomass, and represents an opportunity to develop, test, and optimize the necessary technologies to make microalgal biofuels more cost-effective and efficient. However, many constraints on the eventual deployment of this technology must be taken into consideration and mitigating strategies developed before large scale microalgal cultivation can become a reality. As a strategy for CO2 biomitigation from industrial point source emitters, microalgal cultivation can be limited by the availability of land, light, and other nutrients like N and P. Effective removal of N and P from municipal wastewater is limited by the processing capacity of available microalgal cultivation systems. Strategies to mitigate against the constraints are discussed.
Keywords: Microalgae biofuels; Biomass; Wastewater; Flue gas

Erratum to: Integration of microalgae cultivation with industrial waste remediation for biofuel and bioenergy production: opportunities and limitations by Patrick J. McGinn; Kathryn E. Dickinson; Shabana Bhatti; Jean-Claude Frigon; Serge R. Guiot; Stephen J. B. O’Leary (249-249).

The fluxes of CO2 and oxygen during photosynthesis by cell suspensions of Tessellaria volvocina and Mallomonas papillosa were monitored mass spectrometrically. There was no rapid uptake of CO2, only a slow drawdown to compensation concentrations of 26 μM for T. volvocina and 18 μM for M. papillosa, when O2 evolution ceased, indicating a lack of active bicarbonate uptake by the cells. Darkening of the cells after a period of photosynthesis did not cause rapid release of CO2, indicating the absence of an intracellular inorganic carbon pool. However, upon darkening a brief burst of CO2 was observed similar to the post-illumination burst characteristic of C3 higher plants. Treatment of the cells of both species with the membrane-permeable carbonic anhydrase inhibitor ethoxyzolamide had no adverse effect on photosynthetic rate, but stimulated the dark CO2 burst indicating the dark oxidation of a compound formed in the light. In the absence of any active accumulation of inorganic carbon photosynthesis in these species should be inhibited by O2. This was investigated in four synurophyte species T. volvocina, M. papillosa, Synura petersenii, and Synura uvella: photosynthetic O2 evolution rates in all four algae, measured by O2 electrode, were significantly higher (40–50%) in media at low O2 (4%) than in air-equilibrated (21% O2) media, indicating an O2 inhibition of photosynthesis (Warburg effect) and thus the occurrence of photorespiration in these species.
Keywords: Carbon concentrating mechanism; Chrysophyte; Mallomonas ; Mass spectrometry; Photosynthesis; Photorespiration; Synura ; Tessellaria

Most of the experimental work on the effects of ocean acidification on the photosynthesis of algae has been performed in the laboratory using monospecific cultures. It is frequently assumed that the information obtained from these cultures can be used to predict the acclimation response in the natural environment. CO2 concentration is known to regulate the expression and functioning of the CCMs in the natural communities; however, ambient CO2 can become quite variable in the marine ecosystems even in the short- to mid-term. We propose that the degree of saturation of the photosynthesis for a given algal community should be defined in relation to the particular characteristics of its habitat, and not only in relation to its taxonomic composition. The convenience of high CO2 experiments to infer the degree of photosynthesis saturation by CO2 in the natural algal communities under the present ocean conditions, as well as its trend in a coming future is discussed taking into account other factors such as the availability of light and nutrients, and seasonality.
Keywords: Carbonic anhydrase; Ocean acidification; Primary production; Carbonate system; Phytoplankton; Seaweeds

Inorganic carbon can be in short supply in freshwater relative to that needed by freshwater plants for photosynthesis because of a large external transport limitation coupled with frequent depleted concentrations of CO2 and elevated concentrations of O2. Freshwater plants have evolved a host of avoidance, exploitation and amelioration strategies to cope with the low and variable supply of inorganic carbon in water. Avoidance strategies rely on the spatial variation in CO2 concentrations within and among lakes. Exploitation strategies involve anatomical and morphological features that take advantage of sources of CO2 outside of the water column such as the atmosphere or sediment. Amelioration strategies involve carbon-concentrating mechanisms based on uptake of bicarbonate, which is widespread, C4-fixation, which is infrequent, and crassulacean acid metabolism (CAM), which is of intermediate frequency. CAM enables aquatic plants to take up inorganic carbon in the night. Furthermore, daytime inorganic carbon uptake is generally not inhibited and therefore CAM is considered to be a carbon-conserving mechanism. CAM in aquatic plants is a plastic mechanism regulated by environmental variables and is generally downregulated when inorganic carbon does not limit photosynthesis. CAM is regulated in the long term (acclimation during growth), but is also affected by environmental conditions in the short term (response on a daily basis). In aquatic plants, CAM appears to be an ecologically important mechanism for increasing inorganic carbon uptake, because the in situ contribution from CAM to the C-budget generally is high (18–55%).
Keywords: Acclimation; CO2 ; Carbon-concentrating mechanism (CCM); Elodeids; Inorganic carbon; Isoetids; Macrophytes; Regulation

Algal and aquatic plant carbon concentrating mechanisms in relation to environmental change by John A. Raven; Mario Giordano; John Beardall; Stephen C. Maberly (281-296).
Carbon dioxide concentrating mechanisms (also known as inorganic carbon concentrating mechanisms; both abbreviated as CCMs) presumably evolved under conditions of low CO2 availability. However, the timing of their origin is unclear since there are no sound estimates from molecular clocks, and even if there were, there are no proxies for the functioning of CCMs. Accordingly, we cannot use previous episodes of high CO2 (e.g. the Palaeocene–Eocene Thermal Maximum) to indicate how organisms with CCMs responded. Present and predicted environmental change in terms of increased CO2 and temperature are leading to increased CO2 and HCO3 and decreased CO3 2− and pH in surface seawater, as well as decreasing the depth of the upper mixed layer and increasing the degree of isolation of this layer with respect to nutrient flux from deeper waters. The outcome of these forcing factors is to increase the availability of inorganic carbon, photosynthetic active radiation (PAR) and ultraviolet B radiation (UVB) to aquatic photolithotrophs and to decrease the supply of the nutrients (combined) nitrogen and phosphorus and of any non-aeolian iron. The influence of these variations on CCM expression has been examined to varying degrees as acclimation by extant organisms. Increased PAR increases CCM expression in terms of CO2 affinity, whilst increased UVB has a range of effects in the organisms examined; little relevant information is available on increased temperature. Decreased combined nitrogen supply generally increases CO2 affinity, decreased iron availability increases CO2 affinity, and decreased phosphorus supply has varying effects on the organisms examined. There are few data sets showing interactions amongst the observed changes, and even less information on genetic (adaptation) changes in response to the forcing factors. In freshwaters, changes in phytoplankton species composition may alter with environmental change with consequences for frequency of species with or without CCMs. The information available permits less predictive power as to the effect of the forcing factors on CCM expression than for their overall effects on growth. CCMs are currently not part of models as to how global environmental change has altered, and is likely to further alter, algal and aquatic plant primary productivity.
Keywords: CO2 concentrating mechanism; Combined nitrogen; Inorganic carbon; Iron; Mixing depth; Photosynthetically active radiation; Phosphorus; Temperature; UVA–UVB