CO2 exchange characteristics during dark-light transitions in wild-type and mutant Chlamydomonas reinhardii cells

A burst of net CO2 uptake was observed during the first 3–4 min after the onset of illumination in both wild-type Chlamydomonas reinhardii in which carbonic anhydrase was chemically inhibited with ethoxyzolamide and in a mutant of C. reinhardii (ca-1-12-1C) deficient in carbonic anhydrase activity. The burst was followed by a rapid decrease in the CO2 uptake rate so that net evolution often occurred. After a 2–3 min period of CO2 evolution, net CO2 uptake again increased and ultimately reached a steady-state, positive rate. From [14CO2]-tracer studies it was determined that CO2 fixation proceeded at a nearly linear rate throughout the period of illumination. Thus, prior to reaching a steady state, there was a rapid accumulation of inorganic carbon inside the cells which apparently reached a supercritical concentration and the excess was excreted, causing a subsequent efflux of CO2. A post illumination burst of net CO2 efflux was also observed in ethoxyzolamide-inhibited wild type and ca-1 mutant cells, but not in the unihibited wild type. [14CO2]-tracer experiments revealed that this burst was the result of a collapse of a large internal inorganic carbon pool at the onset of darkness rather than a photorespiratory post-illumination burst. These results indicate that upon illumination, chemical or genetic inhibition of carbonic anhydrase initially causes an accumulation of excess inroganic carbon in C. reinhardii cells, and that unknown regulatory mechanisms correct for this imbalance by first excreting the excess inorganic carbon and then, after several dampened oscillations, achieving an equilibrium between bicarbonate uptake, bicarbonate dehydration, and CO2 fixation

CO2 concentrating mechanisms in eukaryotic microalgae

Many aquatic photosynthetic microorganisms possess inducible CO2 concentrating mechanisms (CCMs) that allow them to optimize carbon acquisition in environments with frequently changing and often limiting CO2 concentrations. The CCMs function by accumulation of a large quantity of intracellular inorganic carbon (Ci) through concerted Ci uptake systems and enzymes catalyzing the interconversion between different species of Ci. In addition, an array of regulatory devices appears present to facilitate the sensing of CO2 availability and the regulation of metabolic pathways. Over the past several decades, significant advances have been made in understanding the CCM and its regulation. With the aid of mutant studies and the availability of several cyanobacterial and eukaryotic algal genomes, an integrated picture is emerging to reveal many of the molecular details in the microalgal CCMs. This review will focus on the recent advances in identifying and characterizing the major components involved in the CCM, including Ci uptake systems and regulatory pathways in eukaryotic microalgae, especially in the model organism, Chlamydomonas reinhardtii

Acclimation to very-low CO2: Contribution of LCIB and LCIA to inorganic carbon uptake in Chlamydomonas reinhardtii

The limiting-CO2 inducible CO2 concentrating mechanism (CCM) of microalgae represents an effective strategy to capture CO2 when its availability is limited. At least two limiting-CO2 acclimation states, termed low CO2 and very-low CO2, have been demonstrated in the model microalga Chlamydomonas reinhardtii, and many questions still remain unanswered regarding both the regulation of these acclimation states and the molecular mechanism underlying operation of the CCM in these two states. This study examines the role of two proteins, LCIA (also named NAR1.2) and LCIB, in the CCM of C. reinhardtii. The identification of an LCIA-LCIB double mutant based on its inability to survive in very-low CO2 suggests that both LCIA and LCIB are critical for survival in very-low CO2. The contrasting impacts of individual mutations in LCIB and LCIA in comparison with the impacts of LCIB-LCIA double mutations on growth and Ci-dependent photosynthetic O2 evolution reveal distinct roles of LCIA and LCIB in the CCM. While both LCIA and LCIB are essential for very-low CO2 acclimation, LCIB appears to function in a CO2 uptake system, while LCIA appears to be associated with a HCO3- transport system. The contrasting and complementary roles of LCIA and LCIB in acclimation to low CO2 and very-low CO2 suggest a possible mechanism of differential regulation of the CCM based on the inhibition of HCO3- transporters by moderate to high levels of CO2

A model of carbon dioxide assimilation in Chlamydomonas reinhardii

A simple model of photosynthetic CO2 assimilation in Chlamydomonas has been developed in order to evaluate whether a CO2-concentrating system could explain the photosynthetic characteristics of this alga (high apparent affinity for CO2, low photorespiration, little O2inhibition of photosynthesis, and low CO2 compensation concentration). Similarly, the model was developed to evaluate whether the proposed defects in the CO2-concentrating system of two Chlamydomonas mutants were consistent with their observed photosynthetic characteristics. The model treats a Chlamydomonas cell as a single compartment with two carbon inputs: passive diffusion of CO2, and active transport of HCO3-. Internal inorganic carbon was considered to have two potential fates: assimilation to fixed carbon via ribulose 1,5-bisphosphate carboxylase-oxygenase or exiting the cell by either passive CO2 diffusion or reversal of HCO3-transport. Published values for kinetic parameters were used where possible. The model accurately reproduced the CO2-response curves of photosynthesis for wild-type Chlamydomonas, the two mutants defective in the CO2-concentrating system, and a double mutant constructed by crossing these two mutants. The model also predicts steady-state internal inorganic-carbon concentrations in reasonable agreement with measured values in all four cases. Carbon dioxide compensation concentrations for wild-type Chlamydomonas were accurately predicted by the model and those predicted for the mutants were in qualitative agreement with measured values. The model also allowed calculation of approximate energy costs of the CO2-concentrating system. These calculations indicate that the system may be no more energy-costly than C4 photosynthesis

Use of Mutants in Analysis of the CO2-Concentrating Pathway of Chlamydomonas Reinhardtii

In Chlamydomonas reinhardtii and other green algae, a pathway which actively concentrates CO2 at the site of ribulose 1,5-bisphosphate-carbozylase/oxygenase (RUBISCO) is responsible for the suppression of photo-respiration and oxygen inhibition of photosynthesis and for the stimulation of photosynthesis at a low external CO2 concentrations. Increased photosynthesis and reduced photorepiration in Chlamydomonas at air levels of CO2 and O2 are manifested by a high affinity for CO2 in photosynthesis, a nearly maximal photosynthetic rate, absence of O2 inhibition of CO2 fixation, low rates of synthesis of photo respiratory metabolites, and a near-zero CO2 compensation concentration (1,2). The CO2-concentrating pathway of Chlamydomonas is inducible, with induction occurring at air levels of CO2 but not at elevated (1-5%) concentrations of CO2 (2). Biochemical and physiological studies implicated the involvement of at least two components in the pathway, an energy-dependent, saturable inorganic transport process (1,9) and the enzyme carbonic anhydrase (CA) (1,2). Badger et al. (1) suggested that the role of CA in the pathway might be dehydration of transported HCO3 to supple CO2, the substrate of RUBISCO. In order to further characterize the Chlamydomonas CO2-concentrating pathway, we utilized existing, nonphotosynthetic mutants of C. rienhardtii fo study of induction requirements and set out to identify and characterize new mutant strains of C. reinhardtii with defects in the CO2-concentrating pathway itself. This work with Chlamydomonas mutants has helped firmly establish the requirement for photosynthetic competence in the induction of the pathway, unambiguously confirmed that at least two components, CA and HCO3 transport, are involved, and that the principal role of internal CA is dehydration of transported HCO3

Opportunistic proteolytic processing of carbonic anhydrase 1 from Chlamydomonas in Arabidopsis reveals a novel route for protein maturation

Proteolytic processing of secretory proteins to yield an active form generally involves specific proteolytic cleavage of a pre-protein. Multiple specific proteases have been identified that target specific pre-protein processing sites in animals. However, characterization of site-specific proteolysis of plant pre-proteins is still evolving. In this study, we characterized proteolytic processing of Chlamydomonas periplasmic carbonic anhydrase 1 (CAH1) in Arabidopsis. CAH1 pre-protein undergoes extensive post-translational modification in the endomembrane system, including glycosylation, disulfide bond formation and proteolytic removal of a peptide ‘spacer’ region, resulting in a mature, heterotetrameric enzyme with two large and two small subunits. We generated a series of small-scale and large-scale modifications to the spacer and flanking regions to identify potential protease target motifs. Surprisingly, we found that the endoproteolytic removal of the spacer from the CAH1 pre-protein proceeded via an opportunistic process apparently followed by further maturation via amino and carboxy peptidases. We also discovered that the spacer itself is not required for processing, which appears to be dependent only on the number of amino acids separating two key disulfide-bond-forming cysteines. Our data suggest a novel, opportunistic route for pre-protein processing of CAH1

Acclimation of Chlamydomonas to changing carbon availability

Aquatic organisms, including Chlamydomonas reinhardtii, are faced with a variable supply of dissolved inorganic carbon (Ci). Accordingly, C. reinhardtii has the ability to acclimate to the changing Ci supply through a variety of responses, including induction of a CO2 concentrating mechanism (CCM) when Ci is limiting. The CCM uses active Ci uptake to accumulate a high internal concentration of bicarbonate, which is dehydrated by a specific thylakoid carbonic anhydrase to supply CO2, the substrate used in photosynthesis. In addition to the changes demonstrably related to the function of the CCM, C. reinhardtii exhibits several other acclimation responses to limiting Ci, such as changes in cellular organization and induction or upregulation of several genes. A key area currently under investigation is how C. reinhardtii cells recognize the change in Ci or CO2 concentration, and transduce that signal into needed gene expression changes. Mutational analyses are proving very useful for learning more about the CCM and about the acclimation response to changes in Ci availability. Cloning of the gene disrupted in cia5, a mutant apparently unable to acclimate to limiting Ci, has opened opportunities for more rapid progress in understanding the signal transduction pathway. The Cia5 gene appears to encode a transcription factor that may control, either directly or indirectly, much of the gene expression responses to limiting Ci in C. reinhardtii. Several additional new mutants with potential defects in the signal transduction pathway have been isolated, including three new alleles of cia5

The Plastid Casein Kinase 2 Phosphorylates Rubisco Activase at the Thr-78 Site but Is Not Essential for Regulation of Rubisco Activation State

Rubisco activase (RCA) is essential for the activation of Rubisco, the carboxylating enzyme of photosynthesis. In Arabidopsis, RCA is composed of a large RCAα and small RCAβ isoform that are formed by alternative splicing of a single gene (At2g39730). The activity of Rubisco is controlled in response to changes in irradiance by regulation of RCA activity, which is known to involve a redox-sensitive disulfide bond located in the carboxy-terminal extension of the RCAα subunit. Additionally, phosphorylation of RCA threonine-78 (Thr-78) has been reported to occur in the dark suggesting that phosphorylation may also be associated with dark-inactivation of RCA and deactivation of Rubisco. In the present study, we developed site-specific antibodies to monitor phosphorylation of RCA at the Thr-78 site and used non-reducing SDS-PAGE to monitor the redox status of the RCAα subunit. By immunoblotting, phosphorylation of both RCA isoforms occurred at low light and in the dark and feeding peroxide or DTT to leaf segments indicated that redox status of the chloroplast stroma was a critical factor controlling RCA phosphorylation. Use of a knockout mutant identified the plastid-targeted casein kinase 2 (cpCK2α) as the major protein kinase involved in RCA phosphorylation. Studies with recombinant cpCK2α and synthetic peptide substrates identified acidic residues at the –1, +2, and +3 positions surrounding Thr-78 as strong positive recognition elements. The cpck2 knockout mutant had strongly reduced phosphorylation at the Thr-78 site but was similar to wild type plants in terms of induction kinetics of photosynthesis following transfer from darkness or low light to high light, suggesting that if phosphorylation of RCA Thr-78 plays a direct role it would be redundant to redox regulation for control of Rubisco activation state under normal conditions