CO2-dependent migration and relocation of LCIB,a pyrenoid-peripheral protein in Chlamydomonas reinhardtii

Most microalgae overcome the difficulty of acquiring inorganic carbon (Ci) in aquatic environments by inducing a CO₂-concentrating mechanism (CCM). In the green alga Chlamydomonas reinhardtii, two distinct photosynthetic acclimation states have been described under CO₂-limiting conditions (low-CO₂ [LC] and very low-CO₂ [VLC]). LC-inducible protein B (LCIB), structurally characterized as carbonic anhydrase, localizes in the chloroplast stroma under CO₂-supplied and LC conditions. In VLC conditions, it migrates to aggregate around the pyrenoid, where the CO₂-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase is enriched. Although the physiological importance of LCIB localization changes in the chloroplast has been shown, factors necessary for the localization changes remain uncertain. Here, we examined the effect of pH, light availability, photosynthetic electron flow, and protein synthesis on the localization changes, along with measuring Ci concentrations. LCIB dispersed or localized in the basal region of the chloroplast stroma at 8.3–15 µM CO₂, whereas LCIB migrated toward the pyrenoid at 6.5 µM CO₂. Furthermore, LCIB relocated toward the pyrenoid at 2.6–3.4 µM CO₂, even in cells in the dark or treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea and cycloheximide in light. In contrast, in the mutant lacking CCM1, a master regulator of CCM, LCIB remained dispersed even at 4.3 µM CO₂. Meanwhile, a simultaneous expression of LCIC, an interacting protein of LCIB, induced the localization of several speckled structures at the pyrenoid periphery. These results suggest that the localization changes of LCIB require LCIC and are controlled by CO₂ concentration with ∼7 µM as the boundary.

Algal chloroplast proteins undergo localization changes in response to CO2 concentrations, reflecting their physiological function in survival under fluctuating CO2 environments.     

LCIB in the Chlamydomonas CO2-concentrating mechanism

The CO₂-concentrating mechanism confers microalgae a versatile and efficient strategy for adapting to a wide range of environmental CO₂ concentrations. LCIB, which has been demonstrated as a key player in the eukaryotic algal CO₂-concentrating mechanism (CCM), is a novel protein in Chlamydomonas lacking any recognizable domain or motif, and its exact function in the CCM has not been clearly defined. The unique air-dier growth phenotype and photosynthetic characteristics in the LCIB mutants, and re-localization of LCIB between different subcellular locations in response to different levels of CO₂, have indicated that the function of LCIB is closely associated with a distinct low CO₂ acclimation state. Here, we review physiological and molecular evidence linking LCIB with inorganic carbon accumulation in the CCM and discuss the proposed function of LCIB in several inorganic carbon uptake/accumulation pathways. Several new molecular characteristics of LCIB also are presented.     

High-resolution suborganellar localization of Ca<Superscript>2+</Superscript>-binding protein CAS,a novel regulator of CO<Subscript>2</Subscript>-concentrating mechanism

Many aquatic algae induce a CO₂-concentrating mechanism (CCM) associated with active inorganic carbon transport to maintain high photosynthetic affinity using dissolved inorganic carbon even in low-CO₂ (LC) conditions. In the green alga Chlamydomonas reinhardtii, a Ca²⁺-binding protein CAS was identified as a novel factor regulating the expression of CCM-related proteins including bicarbonate transporters. Although previous studies revealed that CAS associates with the thylakoid membrane and changes its localization in response to CO₂ and light availability, its detailed localization in the chloroplast has not been examined in vivo. In this study, high-resolution fluorescence images of CAS fused with a Chlamydomonas-adapted fluorescence protein, Clover, were obtained by using a sensitive hybrid detector and an image deconvolution method. In high-CO₂ (5% v/v) conditions, the fluorescence signals of Clover displayed a mesh-like structure in the chloroplast and part of the signals discontinuously overlapped with chlorophyll autofluorescence. The fluorescence signals gathered inside the pyrenoid as a distinct wheel-like structure at 2 h after transfer to LC-light condition, and then localized to the center of the pyrenoid at 12 h. These results suggest that CAS could move in the chloroplast along the thylakoid membrane in response to lowering CO₂ and gather inside the pyrenoid during the operation of the CCM.     

Thylakoid Lumen Carbonic Anhydrase (CAH3) Mutation Suppresses Air-Dier Phenotype of LCIB Mutant in Chlamydomonas reinhardtii

An active CO₂-concentrating mechanism is induced when Chlamydomonas reinhardtii acclimates to limiting inorganic carbon (Ci), either low-CO₂ (L-CO₂; air level; approximately 0.04% CO₂) or very low-CO₂ (VL-CO₂; approximately 0.01% CO₂) conditions. A mutant, ad1, which is defective in the limiting-CO₂-inducible, plastid-localized LCIB, can grow in high-CO₂ or VL-CO₂ conditions but dies in L-CO₂, indicating a deficiency in a L-CO₂-specific Ci uptake and accumulation system. In this study, we identified two ad1 suppressors that can grow in L-CO₂ but die in VL-CO₂. Molecular analyses revealed that both suppressors have mutations in the CAH3 gene, which encodes a thylakoid lumen localized carbonic anhydrase. Photosynthetic rates of L-CO₂-acclimated suppressors under acclimation CO₂ concentrations were more than 2-fold higher than ad1, apparently resulting from a more than 20-fold increase in the intracellular concentration of Ci as measured by direct Ci uptake. However, photosynthetic rates of VL-CO₂-acclimated cells under acclimation CO₂ concentrations were too low to support growth in spite of a significantly elevated intracellular Ci concentration. We conclude that LCIB functions downstream of CAH3 in the CO₂-concentrating mechanism and probably plays a role in trapping CO₂ released by CAH3 dehydration of accumulated Ci. Apparently dehydration by the chloroplast stromal carbonic anhydrase CAH6 of the very high internal Ci caused by the defect in CAH3 provides Rubisco sufficient CO₂ to support growth in L-CO₂-acclimated cells, but not in VL-CO₂-acclimated cells, even in the absence of LCIB.CO₂ serves both as the substrate for photosynthesis and as an important signal to regulate plant growth and development, so variable CO₂ concentrations can impact photosynthesis, growth, and productivity of plants. Terrestrial C₄ plants have developed a CO₂-concentrating mechanism (CCM) involving anatomical and biochemical adaptations to accumulate a higher concentration of CO₂ as substrate Rubisco and to suppress oxygenation of ribulose-1,5-bisP, a wasteful side reaction. In contrast, a different type of CCM is induced in the unicellular green microalga Chlamydomonas reinhardtii when the supply of dissolved inorganic carbon (Ci; CO₂ and HCO₃⁻) for photosynthesis is limited (Beardall and Giordano, 2002; Giordano et al., 2005; Moroney and Ynalvez, 2007; Spalding, 2008). In response to limiting CO₂, the CCM uses active Ci transport, both at the plasma membrane and the chloroplast envelope, to accumulate a high concentration of HCO₃⁻ within the chloroplast (Palmqvist et al., 1988; Sültemeyer et al., 1988). The thylakoid lumen carbonic anhydrase (CAH3) plays an essential role in the rapid dehydration of the accumulated HCO₃⁻ to release CO₂ into the pyrenoid, a Rubisco-containing internal compartment of the chloroplast, for assimilation by Rubisco (Price et al., 2002; Spalding et al., 2002).While a number of genes and proteins essential to the operation of the CCM in C. reinhardtii have been identified, our understanding of Ci uptake and its regulation, as well as other aspects of CCM function is limited. A better understanding of the similar CCM in prokaryotic organisms, specifically the cyanobacteria Synechocystis and Synechococcus, has been gained. At least five different types of Ci transporters have been identified in cyanobacteria, including three HCO₃⁻ transporters and two active CO₂ uptake systems (Price et al., 2002, 2004).Recently, at least three distinct CO₂-regulated acclimation states were identified in C. reinhardtii based on growth, photosynthesis and gene expression characteristics, a high-CO₂ (H-CO₂) state (5%–0.5% CO₂), low-CO₂ (L-CO₂) state (air level; 0.4%–0.03% CO₂), and very low-CO₂ (VL-CO₂) state (0.01%–0.005% CO₂; Vance and Spalding, 2005). Two allelic HCR (H-CO₂-requiring) mutants, pmp1 and ad1, grow as well (pmp1) or nearly as well (ad1) as wild-type cells in both H-CO₂ and VL-CO₂ conditions while only dying in L-CO₂, indicating a deficient Ci transport and/or accumulation system only in the L-CO₂ acclimation state (Spalding et al., 1983b, 2002). The defective gene responsible for the pmp1/ad1 phenotype was identified as LCIB, a limiting CO₂-inducible gene, the product of which is predicted to be located in the chloroplast stroma and proposed to be involved with chloroplast Ci uptake in L-CO₂ conditions (Wang and Spalding, 2006). The LCIB gene product is a member of a small gene family so far only found in a few microalgae species (Spalding, 2008).To investigate the roles of LCIB in eukaryotic photosynthetic organisms and identify other functional components involved in chloroplast Ci accumulation in C. reinhardtii, we used an insertional mutagenesis approach to select suppressors of the air-dier phenotype of the LCIB mutant ad1. In this study, we describe two ad1 suppressors, ad-su6 and ad-su7, that grow normally in L-CO₂ but, unlike ad1, die in VL-CO₂. This report also presents data suggesting that the air-dier phenotype of ad1 is suppressed by increased intracellular Ci concentrations in the two suppressors, and suggesting a possible role for LCIB as a CO₂ trap rather than having any direct role in chloroplast envelope Ci transport.     

The CO2-concentrating mechanism in a starchless mutant of the green unicellular alga Chlorella pyrenoidosa

The CO₂-concentrating mechanism (CCM) was induced in the green unicellular alga Chlorella when cells were transferred from high (5% CO₂) to low (0.03%) CO₂ concentrations. The induction of the CCM correlated with the formation of a starch sheath specifically around the pyrenoid in the chloroplast. With the aim of clarifying whether the starch sheath was involved in the operation of the CCM, we isolated and physiologically characterized a starchless mutant of Chlorella pyrenoidosa, designated as IAA-36. The mutant strain grew as vigorously as the wild type under high and low CO₂ concentrations, continuous light and a 12 h light/12 h dark photoperiod. The CO₂ requirement for half-maximal rates of photosynthesis [K_0.5(CO₂)] decreased from 40 μM to 2–3 μM of CO₂ when both wild type and mutant were switched from high to low CO₂. The high affinity for inorganic carbon indicates that the IAA-36 mutant is able to induce a fully active CCM. Since the mutant does not have the pyrenoid starch sheath, we conclude that the sheath is not involved in the operation of the CCM in Chlorella cells.     

Isolation and characterization of novel high-CO2-requiring mutants of Chlamydomonas reinhardtii

Aquatic microalgae induce a carbon-concentrating mechanism (CCM) to maintain photosynthetic activity in low-CO₂ (LC) conditions. Although the molecular mechanism of the CCM has been investigated using the single-cell green alga Chlamydomonas reinhardtii, and several CCM-related genes have been identified by analyzing high-CO₂ (HC)-requiring mutants, many aspects of the CO₂-signal transduction pathways remain to be elucidated. In this study, we report the isolation of novel HC-requiring mutants defective in the induction of CCM by DNA tagging. Growth rates of 20,000 transformants grown under HC and LC conditions were compared, and three HC-requiring mutants (H24, H82, and P103) were isolated. The photosynthetic CO₂-exchange activities of these mutants were significantly decreased compared with that of wild-type cells, and accumulation of HLA3 and both LCIA and HLA3 were absent in mutants H24 and H82, respectively. Although the insertion of the marker gene and the HC-requiring phenotype were linked in the tetrad progeny of H82, and a calcium-sensing receptor CAS was disrupted by the insertion, exogenous expression of CAS alone could not complement the HC-requiring phenotype.     

New horizons for building pyrenoid-based CO2-concentrating mechanisms in plants to improve yields

Many photosynthetic species have evolved CO₂-concentrating mechanisms (CCMs) to improve the efficiency of CO₂ assimilation by Rubisco and reduce the negative impacts of photorespiration. However, the majority of plants (i.e. C3 plants) lack an active CCM. Thus, engineering a functional heterologous CCM into important C3 crops, such as rice (Oryza sativa) and wheat (Triticum aestivum), has become a key strategic ambition to enhance yield potential. Here, we review recent advances in our understanding of the pyrenoid-based CCM in the model green alga Chlamydomonas reinhardtii and engineering progress in C3 plants. We also discuss recent modeling work that has provided insights into the potential advantages of Rubisco condensation within the pyrenoid and the energetic costs of the Chlamydomonas CCM, which, together, will help to better guide future engineering approaches. Key findings include the potential benefits of Rubisco condensation for carboxylation efficiency and the need for a diffusional barrier around the pyrenoid matrix. We discuss a minimal set of components for the CCM to function and that active bicarbonate import into the chloroplast stroma may not be necessary for a functional pyrenoid-based CCM in planta. Thus, the roadmap for building a pyrenoid-based CCM into plant chloroplasts to enhance the efficiency of photosynthesis now appears clearer with new challenges and opportunities.

Research on pyrenoid formation has led to key advances toward engineering an algal CO₂-concentrating mechanism into C3 land plants, and a recent model predicts an optimized pathway for future work.     

Mitochondrial carbonic anhydrases are needed for optimal photosynthesis at low CO2 levels in Chlamydomonas

Chlamydomonas reinhardtii can grow photosynthetically using CO₂ or in the dark using acetate as the carbon source. In the light in air, the CO₂ concentrating mechanism (CCM) of C. reinhardtii accumulates CO₂, enhancing photosynthesis. A combination of carbonic anhydrases (CAs) and bicarbonate transporters in the CCM of C. reinhardtii increases the CO₂ concentration at Ribulose 1,5-bisphosphate carboxylase oxygenase (Rubisco) in the chloroplast pyrenoid. Previously, CAs important to the CCM have been found in the periplasmic space, surrounding the pyrenoid and inside the thylakoid lumen. Two almost identical mitochondrial CAs, CAH4 and CAH5, are also highly expressed when the CCM is made, but their role in the CCM is not understood. Here, we adopted an RNAi approach to reduce the expression of CAH4 and CAH5 to study their possible physiological functions. RNAi mutants with low expression of CAH4 and CAH5 had impaired rates of photosynthesis under ambient levels of CO₂ (0.04% CO₂ [v/v] in air). These strains were not able to grow at very low CO₂ (<0.02% CO₂ [v/v] in air), and their ability to accumulate inorganic carbon (C_i = CO₂ + HCO3−) was reduced. At low CO₂ concentrations, the CCM is needed to both deliver C_i to Rubisco and to minimize the leak of CO₂ generated by respiration and photorespiration. We hypothesize that CAH4 and CAH5 in the mitochondria convert the CO₂ released from respiration and photorespiration as well as the CO₂ leaked from the chloroplast to HCO3- thus “recapturing” this potentially lost CO₂.

Mitochondrial carbonic anhydrases CAH4 and CAH5 in Chlamydomonas reinhardtii are involved in maintaining optimal photosynthesis.     

The induction of the CO2-concentrating mechanism is correlated with the formation of the starch sheath around the pyrenoid of Chlamydomonas reinhardtii

The pyrenoid is a prominent proteinaceous structure found in the stroma of the chloroplast in unicellular eukaryotic algae, most multicellular algae, and some hornworts. The pyrenoid contains the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase and is sometimes surrounded by a carbohydrate sheath. We have observed in the unicellular green alga Chlamydomonas reinhardtii Dangeard that the pyrenoid starch sheath is formed rapidly in response to a decrease in the CO₂ concentration in the environment. This formation of the starch sheath occurs coincidentally with the induction of the CO₂-concentrating mechanism. Pyrenoid starch-sheath formation is partly inhibited by the presence of acetate in the growth medium under light and low-CO₂ conditions. These growth conditions also partly inhibit the induction of the CO₂-concentrating mechanism. When cells are grown with acetate in the dark, the CO₂-concentrating mechanism is not induced and the pyrenoid starch sheath is not formed even though there is a large accumulation of starch in the chloroplast stroma. These observations indicate that pyrenoid starch-sheath formation correlates with induction of the CO₂-concentrating mechanism under low-CO₂ conditions. We suggest that this ultrastructural reorganization under lowCO₂ conditions plays a role in the CO₂-concentrating mechanism C. reinhardtii as well as in other eukaryotic algae.     

A 38-kilodalton low-CO2-inducible polypeptide is associated with the pyrenoid in Chlorella vulgaris

In the green alga Chlorella vulgaris UAM 101, a CO₂-concentrating mechanism (CCM) is induced when cells are transferred from high (5%) to low (0.03%) CO₂ concentrations. The induction of the CCM is correlated with de-novo synthesis of several polypeptides that remain to be identified. The internal carbonic anhydrase (CA; EC 4.2.1.1) activity increased 6- to 7-fold within 6 h of acclimation to air. When crude homogenates were further separated into soluble and insoluble fractions, nearly all of the CA activity was associated with the membrane fraction. Immunoblot analysis of cell homogenates probed with antibodies raised against the 37-kDa subunit of periplasmic CA of Chlamydomonas reinhardtii showed a cross-reaction with a single 38-kDa polypeptide in both high- and low-CO₂-grown cells. The up-regulation of the expression of the 38-kDa polypeptide was closely correlated with the increase in internal CA activity. Furthermore, its subcellular location was also correlated with the distribution of the activity. Immunoblot analysis of pyrenoid fractions showed that the 38-kDa polypeptide was concentrated in the pyrenoids from low-CO₂-grown cells but was not present in pyrenoids from high-CO₂-grown cells. In addition, immunogold labeling experiments showed that the protein was mainly associated with membranes crossing the pyrenoid, while it was absent from the pyrenoid matrix. These studies have identified a putative intracellular CA polypeptide associated with the pyrenoid in Chlorella vulgaris, suggesting that this structure may play an important role in the operation of the CCM and the acclimation to low CO₂ conditions. Received: 16 July 1997 / Accepted: 26 April 1998     

The Intracellular Localization of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase in Chlamydomonas reinhardtii

The pyrenoid is a proteinaceous structure found in the chloroplast of most unicellular algae. Various studies indicate that ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is present in the pyrenoid, although the fraction of Rubisco localized there remains controversial. Estimates of the amount of Rubisco in the pyrenoid of Chlamydomonas reinhardtii range from 5% to nearly 100%. Using immunolocalization, the amount of Rubisco localized to the pyrenoid or to the chloroplast stroma was estimated for C. reinhardtii cells grown under different conditions. It was observed that the amount of Rubisco in the pyrenoid varied with growth condition; about 40% was in the pyrenoid when the cells were grown under elevated CO₂ and about 90% with ambient CO₂. In addition, it is likely that pyrenoidal Rubisco is active in CO₂ fixation because in vitro activity measurements showed that most of the Rubisco must be active to account for CO₂-fixation rates observed in whole cells. These results are consistent with the idea that the pyrenoid is the site of CO₂ fixation in C. reinhardtii and other unicellular algae containing CO₂-concentrating mechanisms.     

A chloroplast pump model for the CO2 concentrating mechanism in the diatom Phaeodactylum tricornutum

Prior analysis of inorganic carbon (C_i) fluxes in the diatom Phaeodactylum tricornutum has indicated that transport of C_i into the chloroplast from the cytoplasm is the major C_i flux in the cell and the primary driving force for the CO₂ concentrating mechanism (CCM). This flux drives the accumulation of C_i in the chloroplast stroma and generates a CO₂ deficit in the cytoplasm, inducing CO₂ influx into the cell. Here, the “chloroplast pump” model of the CCM in P. tricornutum is formalized and its consistency with data on CO₂ and HCO₃ ^? uptake rates, carbonic anhydrase (CA) activity, intracellular C_i concentration, intracellular pH, and RubisCO characteristics is assessed. The chloroplast pump model can account for the major features of the data. Analysis of photosynthetic and C_i uptake rates as a function of external C_i concentration shows that the model has the most difficulty obtaining sufficiently low cytoplasmic CO₂ concentrations to support observed CO₂ uptake rates at low external C_i concentrations and achieving high rates of photosynthesis. There are multiple ways in which model parameters can be varied, within a plausible range, to match measured rates of photosynthesis and CO₂ uptake. To increase CO₂ uptake rates, CA activity can be increased, kinetic characteristics of the putative chloroplast pump can be enhanced to increase HCO₃ ^? export, or the cytoplasmic pH can be raised. To increase the photosynthetic rate, the permeability of the pyrenoid to CO₂ can be reduced or RubisCO content can be increased.     

A recombineering pipeline to clone large and complex genes in Chlamydomonas

The ability to clone genes has greatly advanced cell and molecular biology research, enabling researchers to generate fluorescent protein fusions for localization and confirm genetic causation by mutant complementation. Most gene cloning is polymerase chain reaction (PCR)�or DNA synthesis-dependent, which can become costly and technically challenging as genes increase in size, particularly if they contain complex regions. This has been a long-standing challenge for the Chlamydomonas reinhardtii research community, as this alga has a high percentage of genes containing complex sequence structures. Here we overcame these challenges by developing a recombineering pipeline for the rapid parallel cloning of genes from a Chlamydomonas bacterial artificial chromosome collection. To generate fluorescent protein fusions for localization, we applied the pipeline at both batch and high-throughput scales to 203 genes related to the Chlamydomonas CO₂ concentrating mechanism (CCM), with an overall cloning success rate of 77%. Cloning success was independent of gene size and complexity, with cloned genes as large as 23 kb. Localization of a subset of CCM targets confirmed previous mass spectrometry data, identified new pyrenoid components, and enabled complementation of mutants. We provide vectors and detailed protocols to facilitate easy adoption of this technology, which we envision will open up new possibilities in algal and plant research.

A high-throughput system was developed to clone large, complex genes at high frequency and perform mutant complementation and protein tagging with a range of fluorophores in Chlamydomonas reinhardtii.     

Introducing an algal carbon‐concentrating mechanism into higher plants: location and incorporation of key components

Many eukaryotic green algae possess biophysical carbon‐concentrating mechanisms (CCMs) that enhance photosynthetic efficiency and thus permit high growth rates at low CO₂ concentrations. They are thus an attractive option for improving productivity in higher plants. In this study, the intracellular locations of ten CCM components in the unicellular green alga Chlamydomonas reinhardtii were confirmed. When expressed in tobacco, all of these components except chloroplastic carbonic anhydrases CAH3 and CAH6 had the same intracellular locations as in Chlamydomonas. CAH6 could be directed to the chloroplast by fusion to an Arabidopsis chloroplast transit peptide. Similarly, the putative inorganic carbon (Ci) transporter LCI1 was directed to the chloroplast from its native location on the plasma membrane. CCP1 and CCP2 proteins, putative Ci transporters previously reported to be in the chloroplast envelope, localized to mitochondria in both Chlamydomonas and tobacco, suggesting that the algal CCM model requires expansion to include a role for mitochondria. For the Ci transporters LCIA and HLA3, membrane location and Ci transport capacity were confirmed by heterologous expression and H¹⁴CO₃^‐ uptake assays in Xenopus oocytes. Both were expressed in Arabidopsis resulting in growth comparable with that of wild‐type plants. We conclude that CCM components from Chlamydomonas can be expressed both transiently (in tobacco) and stably (in Arabidopsis) and retargeted to appropriate locations in higher plant cells. As expression of individual Ci transporters did not enhance Arabidopsis growth, stacking of further CCM components will probably be required to achieve a significant increase in photosynthetic efficiency in this species.     

Model of the carbon concentrating mechanism in chloroplasts of eukaryotic algae

A generic chloroplast-based model for the carbon concentrating mechanism (CCM) in eukaryotic algae is presented. The fine structure of chloroplasts is represented by separate compartments: marginal and bulk stroma, pyrenoid, girdle lamella, bulk thylakoids, and central lamella traversing the pyrenoid. The roles of the individual structural elements of the chloroplast with respect to the CCM and the effect of carbonic anhydrase activity in various compartments are analysed. Hypothetical HCO(-)(3)transport into the acidic thylakoid lumen is adjusted by imposing an optimization principle: a given [CO(2)] at the site of RuBisCO is achieved with minimum energy costs for the CCM. Our model is highly efficient in terms of saturation of RuBisCO carboxylase activity and the affinity of the chloroplast for CO(2), if either a girdle lamella or a pyrenoid is present. The highest efficiency is achieved with a pyrenoid. A eukaryotic CCM is not necessarily associated with accumulation of dissolved inorganic carbon (DIC) as in cyanobacteria. Chloroplasts are categorized into four types corresponding to morphological characteristics of all major algal classes with regard to the presence of pyrenoids, girdle lamellae, and the distribution of CA activity.     

Role of pyrenoids in the CO2-concentrating mechanism: comparative morphology, physiology and molecular phylogenetic analysis of closely related strains of Chlamydomonas and Chloromonas (Volvocales)

The morphology of the pyrenoid and the physiology of the CO₂-concentrating mechanism (CCM) were investigated in Chlamydomonas (Cd.) mutabilis Gerloff UTEX 578, Cd. radiata Deason et Bold UTEX 966, Cd. augustae Skuja UTEX 1969, Cd. macrostellata Lund SAG 72.81, Cd. bipapillata Bourrelly SAG 11-47, and Chloromonas (Cr.) insignis Gerloff et Ettl NIES-447, all of which are closely related phylogenetically to the pyrenoid-less strains of Chloromonas. In the chloroplasts of Cd. mutabilis UTEX 578, Cd. radiata UTEX 966, Cd. augustae UTEX 1969, and Cd. macrostellata SAG 72.81, a typical, spheroidal, electron-dense pyrenoid matrix surrounded by starch granules was present, and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) molecules were highly concentrated in the pyrenoid matrix. On the other hand, while the pyrenoid matrix of Cr. insignis NIES-447 was electron-dense that of Cd. bipapillata SAG 11-47 was not, and neither was surrounded by starch granules. The pyrenoid matrices of these two species exhibited a higher concentration of Rubisco molecules than the thylakoid region (thylakoid and stroma) of the chloroplasts; however, the densities of Rubisco molecules in these pyrenoid matrices were low compared with those of the other four Chlamydomonas strains examined in this study and that of Cd. reinhardtii Dangeard. In all six strains examined, the presence of the CCM was indicated by relatively high photosynthetic affinities for CO₂ (low values of K_0.5(CO₂)). However, differences in the inorganic carbon (Ci) pools were recognized in relation to the differences in pyrenoid morphology among the strains. In the typical pyrenoid-containing strains. Cd. mutabilis UTEX 578 and Cd. radiata UTEX 966, the ratio of internal to external inorganic carbon was about 20, while in Cr. insignis NIES-447 and Cd. bipapillata SAG 11-47 the ratio was only 2–3 similar to the two pyrenoid-less, CCM-containing strains of Chloromonas previously examined (E. Morita et al., 1998, Planta 204: 269–276). It is thus speculated that the presence of typical pyrenoids with a high concentration of Rubisco molecules is related to the formation of large Ci pools in the CCM. Detailed phylogenetic relationships among these Chlamydomonas/Chloromonas strains and the pyrenoid-less Chloromonas strains previously investigated were inferred based on the sequence of rbcL, the gene for the large subunit of Rubisco. Two monophyletic groups were resolved with high bootstrap values. Based on the tree topology resolved, it was inferred that loss of the typical pyrenoids accompanied by a decrease in intracellular Ci pools might have taken place independently in the two groups. Received: 21 August 1998 / Accepted: 30 November 1998     

Distribution and functional role of carbonic anhydrase Cah3 associated with thylakoid membranes in the chloroplast and pyrenoid of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

Localization of lumenal carbonic anhydrase Cah3 in thylakoid membranes of Chlamydomonas reinhardtii was studied using wild-type algae and photosynthetic mutants with different composition of chlorophyll-protein complexes in the photosystems. In addition, the photosynthetic characteristics of wild-type C. reinhardtii and cia3 mutants lacking the activity of carbonic anhydrase Cah3 were examined. Western blot analysis revealed the lack of cross reaction with antibodies to Cah3 in the mutant lacking the photosystem II (PSII) reaction center, in contrast to the mutant deficient in light-harvesting complex of PSII. These data show that the lumenal Cah3 is associated with polypeptides on the donor side of PSII reaction center. Using immunoelectron microscopy and antibodies to Cah3 from C. reinhardtii, we showed for the first time that the major part of thylakoid Cah3 is localized in the pyrenoid where the bulk of Rubisco is located. The rate of photosynthetic oxygen evolution and PSII photochemical efficiency were lower in C. reinhardtii cia3 mutant than in the wild type, especially in the cells grown at limiting CO₂ concentrations. These observations show that Cah3 takes part in CO₂-concentrating mechanism of the chloroplast. The results support our hypothesis [1, 2] that the carboxylation reaction in microalgae proceeds in the pyrenoid, a specific Rubisco-containing part of the chloroplast, which acquires CO₂ from the lumen of intrapyrenoid thylakoids. We discuss significance of the pyrenoid as an autonomous metabolic microcompartment, in which Cah3 plays a key role in the production and concentration of CO₂ for Rubisco. These functions may promote the photosynthetic efficiency owing to the effective CO₂ supply for the Calvin cycle.