Photosynthetic Affinity For Inorganic Carbon Research Articles

The Chlamydomonas bZIP transcription factor BLZ8 confers oxidative stress tolerance by inducing the carbon-concentrating mechanism.

Photosynthetic organisms are exposed to various environmental sources of oxidative stress. Land plants have diverse mechanisms to withstand oxidative stress, but how microalgae do so remains unclear. Here, we characterized the Chlamydomonas reinhardtii basic leucine zipper (bZIP) transcription factor BLZ8, which is highly induced by oxidative stress. Oxidative stress tolerance increased with increasing BLZ8 expression levels. BLZ8 regulated the expression of genes likely involved in the carbon-concentrating mechanism (CCM): HIGH-LIGHT ACTIVATED 3 (HLA3), CARBONIC ANHYDRASE 7 (CAH7), and CARBONIC ANHYDRASE 8 (CAH8). BLZ8 expression increased the photosynthetic affinity for inorganic carbon under alkaline stress conditions, suggesting that BLZ8 induces the CCM. BLZ8 expression also increased the photosynthetic linear electron transfer rate, reducing the excitation pressure of the photosynthetic electron transport chain and in turn suppressing reactive oxygen species (ROS) production under oxidative stress conditions. A carbonic anhydrase inhibitor, ethoxzolamide, abolished the enhanced tolerance to alkaline stress conferred by BLZ8 overexpression. BLZ8 directly regulated the expression of the three target genes and required bZIP2 as a dimerization partner in activating CAH8 and HLA3. Our results suggest that a CCM-mediated increase in the CO2 supply for photosynthesis is critical to minimize oxidative damage in microalgae, since slow gas diffusion in aqueous environments limits CO2 availability for photosynthesis, which can trigger ROS formation.

Dynamics of carbon-concentrating mechanism induction and protein relocalization during the dark-to-light transition in synchronized Chlamydomonas reinhardtii.

In the model green alga Chlamydomonas reinhardtii, a carbon-concentrating mechanism (CCM) is induced under low CO2 in the light and comprises active inorganic carbon transport components, carbonic anhydrases, and aggregation of Rubisco in the chloroplast pyrenoid. Previous studies have focused predominantly on asynchronous cultures of cells grown under low versus high CO2. Here, we have investigated the dynamics of CCM activation in synchronized cells grown in dark/light cycles compared with induction under low CO2. The specific focus was to undertake detailed time course experiments comparing physiology and gene expression during the dark-to-light transition. First, the CCM could be fully induced 1 h before dawn, as measured by the photosynthetic affinity for inorganic carbon. This occurred in advance of maximum gene transcription and protein accumulation and contrasted with the coordinated induction observed under low CO2. Between 2 and 1 h before dawn, the proportion of Rubisco and the thylakoid lumen carbonic anhydrase in the pyrenoid rose substantially, coincident with increased CCM activity. Thus, other mechanisms are likely to activate the CCM before dawn, independent of gene transcription of known CCM components. Furthermore, this study highlights the value of using synchronized cells during the dark-to-light transition as an alternative means of investigating CCM induction.

Adaptation of a Chlamydomonas mutant with reduced rate of photorespiration to different concentrations of CO2

It has been widely accepted that Chlamydomonas reinhardtii cells utilize inorganic carbon very efficiently for photosynthesis by operating a CO2-concentrating mechanism (CCM) under conditions of limited CO2. To help define the mechanism, 7FR2N, one of the suppressor double mutants of phosphoglycolate phosphatase-deficient (pgp1) mutants that have a reduced photorespiration rate (RPR) was crossed with wild-type strains to generate the strain N21 as a single RPR mutant. The comparison of photosynthetic characteristics with wild-type strains after the cells adapted to different concentrations of CO2 revealed that photosynthetic affinity for inorganic carbon was higher than that in wild-type strains after adaptation to concentrations between 50 µL·L–1 CO2 and 5% CO2. Chlorophyll fluorescence parameters were also compared, and the biggest difference between N21 and the wild-type strains was observed in the photochemical quenching and effective quantum yield of photosystem II (ΔF/Fm′) at the CO2 compensation point. These values in N21 increased in a similar manner to the photosynthetic affinity for CO2, and increased significantly when the cells adapted to low-CO2 levels, whereas the values in the wild-type strains were apparently lower without any significant changes, regardless of the CO2 concentrations to which they were adapted. Although it was not clear if a nonphotochemical quenching parameter (NPQ) in N21 was higher than that in wild-type strains, NPQ increased coincidentally with the increase in photosynthetic affinity for inorganic carbon when the CO2 concentrations to which the strains were adapted decreased, in both the mutant and wild-type strain, suggesting that this form of NPQ reflects the operation of CCM in certain conditions. Possible candidates for the RPR mutation and the relationship between CCM and photosynthetic electron flow are discussed.Key words: Chlamydomonas reinhardtii, chlorophyll fluorescence, CO2-concentrating mechanism, low-CO2 responsive gene, phosphoglycolate phosphatase, photorespiration.

The novel Myb transcription factor LCR1 regulates the CO2-responsive gene Cah1, encoding a periplasmic carbonic anhydrase in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii acclimates to CO2-limiting stress by inducing a set of genes for a carbon-concentrating mechanism (CCM). This set includes the gene Cah1, which encodes a periplasmic carbonic anhydrase. Although physiological aspects of CO2response have been extensively studied, regulatory components, such as transcription factors involved in the acclimation, have not been well described in eukaryotic microalgae. Using an arylsulfatase gene driven by the Cah1 promoter, a regulatory mutant of Cah1 was isolated and named lcr1 (for low-CO2 stress response). The photosynthetic affinity for inorganic carbon of lcr1 was reduced compared with that of wild-type cells. Expression of three low-CO2-inducible genes, Cah1, Lci1, and Lci6, were regulated by LCR1 as shown by cDNA array and RNA gel blot analyses. The Lcr1 gene encodes a protein of 602 amino acids containing a single Myb domain, which binds to the Cah1-promoter region. Expression of Lcr1 was induced by lowering CO2 levels and controlled by the regulatory factor CCM1. These results suggest that LCR1 transmits the low CO2 signal to at least three CO2-responsive genes and then fully induces CCM.

ISOLATION OF ccmKLMN GENES FROM THE MARINE CYANOBACTERIUM, SYNECHOCOCCUS SP. PCC7002 (CYANOPHYCEAE), AND EVIDENCE THAT CcmM IS ESSENTIAL FOR CARBOXYSOME ASSEMBLY

A high CO2 requiring mutant of the marine cyanobacterium Synechococcus PCC7002 was generated using a random gene‐tagging procedure. This mutant demonstrated a reduced photosynthetic affinity for inorganic carbon (Ci) and accumulated high internal levels of Ci that could not be used for photosynthesis. Analysis of the mutant genomic DNA showed that the mutagenesis had disrupted a cluster of genes involved in the cyanobacterial CO2 concentrating mechanism (CCM), the so‐called ccm genes. These characteristics are consistent with a cyanobacterial mutant with defects in carboxysome assembly and/or functioning. Further genomic analyses indicated that the genes of the Synechococcus PCC7002 operon, ccmKLMN, are structurally similar to those of two closely related cyanobacteria, Synechococcus PCC7942 and Synechocystis PCC6803. The Synechococcus PCC7002 ccmM gene, which encodes a polypeptide with a predicted size of 70 kDa, was the direct target of the mutagenesis event. The CcmM protein has two distinct regions: an N‐terminal region that shows similarity to an archaeon gamma carbonic anhydrase and a C‐terminal region that contains repeated domains demonstrating sequence similarity to the small subunit of Rubisco. Physiological analysis of a ccmM‐defined mutant showed that these cells were essentially identical to the original mutant; they required high CO2 concentrations for growth, they had a low photosynthetic affinity for Ci, and they internalized Ci to high levels. Moreover, ultrastructural examination showed that both the original and the defined mutants lack carboxysomes. Thus, our results demonstrate that the ccmM gene of Synechococcus PCC7002 encodes a polypeptide that is essential for carboxysome assembly and therefore for proper functioning of the cyanobacterial CCM.

Effect of aminooxyacetate, an inhibitor blocking the glycolate pathway, on the induction of a CO2-concentrating mechanism and low-CO2-inducible polypeptides in Chlamydomonas reinhardtii (Chlorophyta)

Polypeptides of 21, 36 and 37 kDa are induced in the unicellular green alga Chlamydomonas reinhardtii when cells are transferred from high (5%) to low (0·03%) CO2 concentrations. The synthesis of these polypeptides is correlated with the induction of the CO2-concentrating mechanism. Interaction between the induction of low-CO2-inducible polypeptides, the CO2-concentrating mechanism and photorespiration has been studied in wild-type C. reinhardtii with the aim of clarifying whether the glycolate pathway is involved in algal acclimation to limiting CO2 conditions. Our results showed that the induction of the 37 kDa periplasmic carbonic anhydrase and 21 kDa polypeptide under low-CO2 conditions was not observed in the presence of aminooxyacetate, an inhibitor which completely blocks glycolate metabolism. However, the induction of the 36 kDa polypeptide was not affected by this inhibitor. The presence of aminooxyacetate during the acclimation to low CO2 conditions also inhibited the increase in the photosynthetic affinity for inorganic carbon shown by low-CO2-acclimated Chlamydomonas cells without the inhibitor. Our results suggest that the induction of the CO2-concentrating mechanism may require the function of the glycolate pathway. In addition, our results also indicate that there is differential regulation of the induction of these three low-CO2-inducible polypeptides in Chlamydomonas reinhardtii.

Effect of aminooxyacetate, an inhibitor blocking the glycolate pathway, on the induction of a CO2-concentrating mechanism and low-CO2-inducible polypeptides in Chlamydomonas reinhardtii (Chlorophyta)

Polypeptides of 21, 36 and 37 kDa are induced in the unicellular green alga Chlamydomonas reinhardtii when cells are transferred from high (5%) to low (0.03%) CO2 concentrations. The synthesis of these polypeptides is correlated with the induction of the CO2-concentrating mechanism. Interaction between the induction of low-CO2-inducible polypeptides, the CO2-concentrating mechanism and photorespiration has been studied in wild-type C. reinhardtii with the aim of clarifying whether the glycolate pathway is involved in algal acclimation to limiting CO2 conditions. Our results showed that the induction of the 37 kDa periplasmic carbonic anhydrase and 21 kDa polypeptide under low-CO2 conditions was not observed in the presence of aminooxyacetate, an inhibitor which completely blocks glycolate metabolism. However, the induction of the 36 kDa polypeptide was not affected by this inhibitor. The presence of aminooxyacetate during the acclimation to low CO2 conditions also inhibited the increase in the photosynthetic affinity for inorganic carbon shown by low-CO2-acclimated Chlamydomonas cells without the inhibitor. Our results suggest that the induction of the CO2-concentrating mechanism may require the function of the glycolate pathway. In addition, our results also indicate that there is differential regulation of the induction of these three low-CO2-inducible polypeptides in Chlamydomonas reinhardtii.

High CO2 Concentration Alleviates the Block in Photosynthetic Electron Transport in an ndhB-Inactivated Mutant of Synechococcus sp. PCC 7942

The high-concentration CO2-requiring mutant N5 of Synechococcus sp. PCC 7942 was obtained by the insertion of a kanamycin-resistant gene at the EcoRI site, 12.4 kb upstream of rbc. The mutant is unable to accumulate inorganic carbon internally and exhibits very low apparent photosynthetic affinity for inorganic carbon but a photosynthetic Vmax similar to that of the wild type. Sequence and northern analyses showed that the insertion inactivated a gene highly homologous to ndhB, encoding subunit II of NADH dehydrogenase in Synechocystis sp. PCC 6803 (T. Ogawa [1991] Proc Natl Acad Sci USA 88: 4275-4279). When the mutant and the wild-type cells were exposed to 5% CO2 in air, their photosynthetic electron transfer capabilities, as revealed by fluorescence and thermoluminescence measurements, were similar. On the other hand, a significant decrease in variable fluorescence was observed when the mutant (but not the wild-type) cells were exposed to low CO2 under continuous light. The same treatment also resulted in a shift (from 38-27 degrees C) in the temperature at which the maximal thermoluminescence emission signal was obtained in the mutant but not in the wild type. These results may indicate that subunit II of NADH dehydrogenase is essential for the functional operation of the photosynthetic electron transport in Synechococcus under low but not high levels of CO2. We suggest that the inability to accumulate inorganic carbon under air conditions stems from disrupture of electron transport in this mutant.

Phenotypic Complementation of High CO2-Requiring Mutants of the Cyanobacterium Synechococcus sp. Strain PCC 7942 by Inosine 5′-Monophosphate

The high CO(2)-requiring mutants of Synechococcus PCC 7942, D4 and R14, were obtained by deletion or inactivation (respectively) of an open reading frame immediately downstream of rbc (the operon encoding the subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase). These mutants exhibit photosynthetic characteristics similar to those of high CO(2)-grown wild type, unlike other cyanobacterial high CO(2)-requiring mutants, where the apparent photosynthetic affinity for inorganic carbon is approximately 2 orders of magnitude lower than that of the wild type. Sequence analysis and metabolic complementation of the mutants by inosine 5'-monophosphate identified this open reading frame as the cyanobacterial equivalent of purK, the eubacterial gene encoding subunit II of phosphoribosyl aminoimidazole carboxylase in the purine biosynthetic pathway. Exposure of high CO(2)-grown Synechococcus to low CO(2) conditions led to the induction of transcription of purK. It is suggested that the high CO(2)-requiring phenotype of these mutants resulted from the defect in purine biosynthesis after exposure to low CO(2). We also raise the possibility that the level of cellular purines is involved in the process of adaptation of cyanobacteria to low concentrations of CO(2).

Photosynthesis and photorespiration in a mutant of the cyanobacterium Synechocystis PCC 6803 lacking carboxysomes

A mutant of the cyanobacterium Synechocystis PCC 6803 was obtained by replacing the gene of the carboxylation enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) with that of the photosynthetic bacterium Rhodospirillum rubrum. This mutant consequently lacks carboxysomes - the protein complexes in which the original enzyme is packed. It is incapable of growing at atmospheric CO2 levels and has an apparent photosynthetic affinity for inorganic carbon (Ci) which is 1000 times lower than that of the wild type, yet it accumulates more Ci than the wild type. The mutant appears to be defective in its ability to utilize the intracellular Ci pool for photosynthesis. Unlike the carboxysomal carboxylase activity of Rubisco, which is almost insensitive to inhibition by O2 in vitro, the soluble enzyme is competitively inhibited by O2. The photosynthetic rate and Ci compensation point of the wild type were hardly affected by low O2 levels. Above 100 μM O2, however, both parameters became inhibited. The CO2 compensation point of the mutant was linearly dependent on O2 concentration. The higher sensitivity of the mutant to O2 inhibition than that expected from in-vitro kinetics parameters of Rubisco, indicates a low capacity to recycle photorespiratory metabolites to Calvin-cycle intermediates.

The CO2concentrating mechanism in cyanobactiria and microalgae

Over the past 10 years it has become clear that cyanobacteria and microalgae possess mechanisms for actively acquiring inorganic carbon from the external medium and are able to use this to elevate the CO2 concentration around the active site of the primary photosynthetic carboxylating enzyme, ribulose bisphosphate carboxylase‐oxygenase (Rubisco). This results in a vastly enhanced photosynthetic affinity for inorganic carbon (Ci) and improved photosynthetic efficiency. The CO2 concentrating mechanism is dependent on the existence of membrane bound Ci transport systems, and a microenvironment within the cell where the accumulated Ci can be used to elevate CO2 at the site of Rubisco. Evidence presented in this review suggests that in cyanobacteria this is achieved by the packaging of Rubisco and carbonic anhydrase (CA) into discrete structures, which are termed carboxysomes. Analogous structures in microalgae, termed pyrenoids, may perform a similar function. The recovery and analysis of high‐CO2‐requiring mutants has greatly advanced our understanding of the mechanisms and genes underlying these systems, especially in cyanobacteria, and this review places particular emphasis on the contribution made by molecular genetic approaches.

Molecular analysis of high CO2 requiring mutants: involvement of genes in the region of rbc, including rbcS, in the ability of cyanobacteria to grow under low CO2

Modifications of the genomic region near (and including) rbc, the operon that codes for the large and small subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), resulted in cyanobacterial mutants that demand high CO2 for growth. Mutant EK6 originated from the fusion of the 3' end of rbcS (which codes for the small subunit of Rubisco) with 84 nucleotides from the 5′ flanking region of nptII (kanamycin-resistance gene), leading to a 17-kDa small subunit, as compared to 14 kDa in the wild type. Mutant D4 originated from substitution of the 1.4-kb PstI fragment, downstream of rbc, with nptII, inactivating several open reading frames in this region. Mutant O105 was obtained by chemical mutagenesis and the mutation was mapped approximately 9 kb upstream of rbc. Mutants EK6 and O105 exhibited a very low apparent photosynthetic affinity for inorganic carbon, whereas D4 had an affinity similar to that observed in wild-type cells grown under high CO2. These mutants, and the constructs used to raise them, can be used to study the role of the small subunit of Rubisco and the genomic region near rbc in cyanobacterial photosynthesis. We propose that this region contains a cluster of genes involved in the ability of cyanobacteria to grow under low ambient CO2..

A Photorespiratory Mutant of Chlamydomonas reinhardtii

A mutant strain of Chlamydomonas reinhardtii, designated 18-7F, has been isolated and characterized. 18-7F requires a high CO(2) concentration for photoautrophic growth in spite of the apparent induction of a functional CO(2) concentrating mechanism in air-adapted cells. In 2% O(2) the photosynthetic characteristics of 18-7F and wild type are similar. In 21% O(2), photosynthetic O(2) evolution is severely inhibited in the mutant by preillumination in limiting CO(2), although the apparent photosynthetic affinity for inorganic carbon is similar in preilluminated cells and in cells incubated in the dark prior to O(2) evolution measurements. Net CO(2) uptake is also inhibited when the cells are exposed to air (21% O(2), 0.035% CO(2), balance N(2)) for longer than a few minutes. [(14)C]Phosphoglycolate accumulates within 5 minutes of photosynthetic (14)CO(2) fixation in cells of 18-7F. Phosphoglycolate does not accumulate in wild type. Phosphoglycolate phosphatase activity in extracts from air-adapted cells of 18-7F is 10 to 20% of that in wild-type Chlamydomonas. The activity of phosphoglycolate phosphatase in heterozygous diploids is intermediate between that of homozygous mutant and wild-type diploids. It was concluded that the high-CO(2) requiring phenotype in 18-7F results from a phosphoglycolate phosphatase deficiency. Genetic analyses indicated that this deficiency results from a single-gene, nuclear mutation. We have named the locus pgp-1.

Adaptation of Chlamydomonas reinhardtii High-CO2-Requiring Mutants to Limiting CO2

Photosynthetic characteristics of four high-CO(2)-requiring mutants of Chlamydomonas reinhardtii were compared to those of wild type before and after a 24-hour exposure to limiting CO(2) concentrations. The four mutants represent two loci involved in the CO(2)-concentrating system of this unicellular alga. All mutants had a lower photosynthetic affinity for inorganic carbon than did the wild type when grown at an elevated CO(2) concentration, indicating that the genetic lesion in each is expressed even at elevated CO(2) concentrations. Wild type and all four mutants exhibited adaptive responses to limiting CO(2) characteristic of the induction of the CO(2)-concentrating system, resulting in an increased affinity for inorganic carbon only in wild type. Although other components of the CO(2)-concentrating system were induced in these mutants, the defective component in each was sufficient to prevent any increase in the affinity for inorganic carbon. It was concluded that the genes corresponding to the ca-1 and pmp-1 loci exhibit at least partially constitutive expression and that all components of the CO(2)-concentrating system may be required to significantly affect the photosynthetic affinity for inorganic carbon.

A Mutant of Synechococcus PCC7942 Incapable of Adapting to Low CO2 Concentration

Some properties of a mutant (RK1) of Synechococcus PCC7942, which requires high CO(2) for growth, are described. The photosynthetic affinity for inorganic carbon (C(i)) in RK1 was about 40 times lower than that in the wild type (WT) when grown at 3% CO(2) (H-cells) and did not change during 10 hours of exposure to low CO(2) (air containing 0.04% CO(2)). The gas exchange of WT and RK1 cells was measured using an open gas-analysis system. All the measurements were performed at a CO(2) concentration of 400 microliters per liter under the conditions where photosynthetic CO(2) fixation is inhibited. When the suspension of H-cells of WT or RK1 was illuminated, the rate of CO(2) influx from the gas phase into the suspension was low and addition of carbonic anhydrase during illumination released only a small amount of CO(2) from the medium into the gas phase. The rate of CO(2) influx and the amount of CO(2) released by carbonic anhydrase were increased in WT during low CO(2) adaptation. These changes did not occur in RK1 during exposure to low CO(2). Cytoplasmic membrane from H-cells of WT or RK1 contained small amount of 42-kilodalton polypeptide. Exposure of RK1 to low CO(2) did not have significant effect on the amount of 42-kilodalton polypeptide, while the same treatment on WT resulted in a large increase of this polypeptide. The RK1 mutant appears to be defective in its ability to utilize the intracellular C(i) pool for photosynthesis and also to transmit a low CO(2) signal for inducing the functional and compositional changes observed in WT during low CO(2) adaptation.

Growth and Photosynthesis of the Cyanobacterium Synechococcus leopoliensis in HCO3−-Limited Chemostats

Synechococcus leopoliensis was grown in HCO(3) (-)-limited chemostats. Growth at 50% the maximum rate occurred when the inorganic carbon concentration was 10 to 15 micromolar (or 5.6 to 8.4 nanomolar CO(2)). The O(2) to CO(2) ratios during growth were as high as 192,000 to 1. At growth rates below 80% the maximum rate, essentially all the supplied inorganic carbon was converted to organic carbon, and the cells were carbon limited. Carbon-limited cells used HCO(3) (-) rather than CO(2) for growth. They also exhibited a very high photosynthetic affinity for inorganic carbon in short-term experiments. Cells growing at greater than 80% maximum growth rate, in the presence of high dissolved inorganic carbon, were termed carbon sufficient. These cells had photosynthetic affinities that were about 1000-fold lower than HCO(3) (-)-limited cells and also had a reduced capacity for HCO(3) (-) transport. HCO(3) (-)-limited cells are reminiscent of the air-grown cells of batch culture studies while the carbon sufficient cells are reminiscent of high-CO(2) grown cells. However, the low affinity cells of the present study were growing at CO(2) concentrations less than air saturation. This suggests that supranormal levels of CO(2) not required to induce the physiological changes usually ascribed to high CO(2) cells.

The Location of the Transporting System for Inorganic Carbon and the Nature of the Form Translocated inChlamydomonas reinhardtii

The permeability of the plasmalemma of Chlamydomonas reinhardtii cells was increased by treatment with poly-L-lysine or dimethylsulph oxide as indicated by 3-phosphoglyc eric acid dependent O2 evolution. These treatments decreased the ability of the cells to accumulate inorganic carbon internally and hence their photosynthetic affinity for inorganic carbon in the medium. With saturating light and inorganic carbon, the photosynthetic rate was less affected by the poly-L-lysine and dimethylsulphoxide treatments. Thus the poly-L-lysine and dimethylsulphoxide did not alter the activity of the chloroplasts but rather made the intracellular inorganic carbon pool more freely exchangeable with the medium. It is concluded that the transporting system for inorganic carbon is located at the plasmalemma. Treatment with Diamox, an inhibitor of carbonic anhydrase, did not affect photosynthetic rate and accumulation of inorganic carbon when CO2 was supplied but strongly inhibited both parameters when HCOj was supplied. In a mutant of Chlamydomonas reinhardtii lacking a cell wall, carbonic anhydrase leaks to the medium and uptake of inorganic carbon is much faster when CO2 is supplied than when HCO7 is supplied. These results suggest that CO2 rather than HCOj is the inorganic carbon species that is actively translocated across the plasmalemma.