Engineering the cyanobacterial ATP-driven BCT1 bicarbonate transporter for functional targeting to C3 plant chloroplasts.

The ATP-driven bicarbonate transporter 1 (BCT1), a four-component complex in the cyanobacterial CO2-concentrating mechanism, could enhance photosynthetic CO2 assimilation in plant chloroplasts. However, directing its subunits (CmpA, CmpB, CmpC and CmpD) to three chloroplast sub-compartments is highly complex. Investigating BCT1 integration into Nicotiana benthamiana chloroplasts revealed promising targeting strategies using transit peptides from the intermembrane space protein Tic22 for correct CmpA targeting, while the transit peptide of the chloroplastic ABCD2 transporter effectively targeted CmpB to the inner envelope membrane. CmpC and CmpD were targeted to the stroma by RecA and recruited to the inner envelope membrane by CmpB. Despite successful targeting, expression of this complex in CO2-dependent Escherichia coli failed to demonstrate bicarbonate uptake. We then used rational design and directed evolution to generate new BCT1 forms that were constitutively active. Several mutants were recovered, including a CmpCD fusion. Selected mutants were further characterized and stably expressed in Arabidopsis thaliana, but the transformed plants did not have higher carbon assimilation rates or decreased CO2 compensation points in mature leaves. While further analysis is required, this directed evolution and heterologous testing approach presents potential for iterative modification and assessment of CO2-concentrating mechanism components to improve plant photosynthesis.

Carbonic anhydrases in the cell wall and plasma membrane of Arabidopsis thaliana are required for optimal plant growth on low CO2.

Introduction: Plants have many genes encoding both alpha and beta type carbonic anhydrases. Arabidopsis has eight alpha type and six beta type carbonic anhydrase genes. Individual carbonic anhydrases are localized to specific compartments within the plant cell. In this study, we investigate the roles of αCA2 and ßCA4.1 in the growth of the plant Arabidopsis thaliana under different CO2 regimes. Methods: Here, we identified the intracellular location of αCA2 and ßCA4.1 by linking the coding region of each gene to a fluorescent tag. Tissue expression was determined by investigating GUS expression driven by the αCA2 and ßCA4.1 promoters. Finally, the role of these proteins in plant growth and photosynthesis was tested in plants with T-DNA insertions in the αCA2 and ßCA4 genes. Results: Fluorescently tagged proteins showed that αCA2 is localized to the cell wall and ßCA4.1 to the plasma membrane in plant leaves. Both proteins were expressed in roots and shoots. Plants missing either αCA2 or ßCA4 did not show any growth defects under the conditions tested in this study. However, if both αCA2 and ßCA4 were disrupted, plants had a significantly smaller above- ground fresh weight and rosette area than Wild Type (WT) plants when grown at 200 µL L-1 CO2 but not at 400 and 1,000 µL L-1 CO2. Growth of the double mutant plants at 200 µL L-1 CO2 was restoredif either αCA2 or ßCA4.1 was transformed back into the double mutant plants. Discussion: Both the cell wall and plasma membrane CAs, αCA2 and ßCA4.1 had to be knocked down to produce an effect on Arabidopsis growth and only when grown in a CO2 concentration that was significantly below ambient. This indicates that αCA2 and ßCA4.1 have overlapping functions since the growth of lines where only one of these CAs was knocked down was indistinguishable from WT growth. The growth results and cellular locations of the two CAs suggest that together, αCA2 and ßCA4.1 play an important role in the delivery of CO2 and HCO3 - to the plant cell.

LCIB functions as a carbonic anhydrase: evidence from yeast and Arabidopsis carbonic anhydrase knockout mutants.

Chlamydomonas reinhardtii evolved a CO2-concentrating mechanism (CCM) because of the limited CO2 in its natural environment. One critical component of the C. reinhardtii CCM is the limiting CO2 inducible B (LCIB) protein. LCIB is required for acclimation to air levels of CO2. C. reinhardtii cells with a mutated LCIB protein have an 'air-dier' phenotype when grown in low CO2 conditions, meaning they die in air levels of CO2 but can grow in high and very low CO2 conditions. The LCIB protein functions together with its close homolog in C. reinhardtii, limiting CO2 inducible C protein (LCIC), in a hexameric LCIB-LCIC complex. LCIB has been proposed to act as a vectoral carbonic anhydrase (CA) that helps to recapture CO2 that would otherwise leak out of the chloroplast. Although both LCIB and LCIC are structurally similar to ßCAs, their CA activity has not been demonstrated to date. We provide evidence that LCIB is an active CA using a Saccharomyces cerevisiae CA knockout mutant (∆NCE103) and an Arabidopsis thaliana ßCA5 knockout mutant (ßca5). We show that different truncated versions of the LCIB protein complement ∆NCE103, while the full length LCIB protein complements ßca5 plants, so that both the yeast and plant mutants can grow in low CO2 conditions.

The Chlamydomonas reinhardtii chloroplast envelope protein LCIA transports bicarbonate in planta.

LCIA (low CO2-inducible protein A) is a chloroplast envelope protein associated with the CO2-concentrating mechanism of the green alga Chlamydomonas reinhardtii. LCIA is postulated to be a HCO3- channel, but previous studies were unable to show that LCIA was actively transporting bicarbonate in planta. Therefore, LCIA activity was investigated more directly in two heterologous systems: an Escherichia coli mutant (DCAKO) lacking both native carbonic anhydrases and an Arabidopsis mutant (ßca5) missing the plastid carbonic anhydrase ßCA5. Neither DCAKO nor ßca5 can grow in ambient CO2 conditions, as they lack carbonic anhydrase-catalyzed production of the necessary HCO3- concentration for lipid and nucleic acid biosynthesis. Expression of LCIA restored growth in both systems in ambient CO2 conditions, which strongly suggests that LCIA is facilitating HCO3- uptake in each system. To our knowledge, this is the first direct evidence that LCIA moves HCO3- across membranes in bacteria and plants. Furthermore, the ßca5 plant bioassay used in this study is the first system for testing HCO3- transport activity in planta, an experimental breakthrough that will be valuable for future studies aimed at improving the photosynthetic efficiency of crop plants using components from algal CO2-concentrating mechanisms.

Arabidopsis plastid carbonic anhydrase ßCA5 is important for normal plant growth.

Carbonic anhydrases (CAs) are zinc-metalloenzymes that catalyze the interconversion of CO2 and HCO3-. In heterotrophic organisms, CAs provide HCO3- for metabolic pathways requiring a carboxylation step. Arabidopsis (Arabidopsis thaliana) has 14 α- and ß-type CAs, two of which are plastid CAs designated as ßCA1 and ßCA5. To study their physiological properties, we obtained knock-out (KO) lines for ßCA1 (SALK_106570) and ßCA5 (SALK_121932). These mutant lines were confirmed by genomic PCR, RT-PCR, and immunoblotting. While ßca1 KO plants grew normally, growth of ßca5 KO plants was stunted under ambient CO2 conditions of 400 µL L-1; high CO2 conditions (30,000 µL L-1) partially rescued their growth. These results were surprising, as ßCA1 is more abundant than ßCA5 in leaves. However, tissue expression patterns of these genes indicated that ßCA1 is expressed only in shoot tissue, while ßCA5 is expressed throughout the plant. We hypothesize that ßCA5 compensates for loss of ßCA1 but, owing to its expression being limited to leaves, ßCA1 cannot compensate for loss of ßCA5. We also demonstrate that ßCA5 supplies HCO3- required for anaplerotic pathways that take place in plastids, such as fatty acid biosynthesis.

A Rapid Method for Detecting Normal or Modified Plant and Algal Carbonic Anhydrase Activity Using Saccharomyces cerevisiae.

In recent years, researchers have attempted to improve photosynthesis by introducing components from cyanobacterial and algal CO2-concentrating mechanisms (CCMs) into terrestrial C3 plants. For these attempts to succeed, we need to understand the CCM components in more detail, especially carbonic anhydrase (CA) and bicarbonate (HCO3−) transporters. Heterologous complementation systems capable of detecting carbonic anhydrase activity (i.e., catalysis of the pH-dependent interconversion between CO2 and HCO3−) or active HCO3− transport can be of great value in the process of introducing CCM components into terrestrial C3 plants. In this study, we generated a Saccharomyces cerevisiae CA knock-out (ΔNCE103 or ΔCA) that has a high-CO2-dependent phenotype (5% (v/v) CO2 in air). CAs produce HCO3− for anaplerotic pathways in S. cerevisiae; therefore, the unavailability of HCO3− for neutral lipid biosynthesis is a limitation for the growth of ΔCA in ambient levels of CO2 (0.04% (v/v) CO2 in air). ΔCA can be complemented for growth at ambient levels of CO2 by expressing a CA from human red blood cells. ΔCA was also successfully complemented for growth at ambient levels of CO2 through the expression of CAs from Chlamydomonas reinhardtii and Arabidopsis thaliana. The ΔCA strain is also useful for investigating the activity of modified CAs, allowing for quick screening of modified CAs before putting them into the plants. CA activity in the complemented ΔCA strains can be probed using the Wilbur−Anderson assay and by isotope exchange membrane-inlet mass spectrometry (MIMS). Other potential uses for this new ΔCA-based screening system are also discussed.

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 (Ci = CO2 + HCO3-) was reduced. At low CO2 concentrations, the CCM is needed to both deliver Ci 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.

Thylakoid localized bestrophin-like proteins are essential for the CO2 concentrating mechanism of Chlamydomonas reinhardtii.

The green alga Chlamydomonas reinhardtii possesses a CO2 concentrating mechanism (CCM) that helps in successful acclimation to low CO2 conditions. Current models of the CCM postulate that a series of ion transporters bring HCO3- from outside the cell to the thylakoid lumen, where the carbonic anhydrase 3 (CAH3) dehydrates accumulated HCO3- to CO2, raising the CO2 concentration for Ribulose bisphosphate carboxylase/oxygenase (Rubisco). Previously, HCO3- transporters have been identified at both the plasma membrane and the chloroplast envelope, but the transporter thought to be on the thylakoid membrane has not been identified. Three paralogous genes (BST1, BST2, and BST3) belonging to the bestrophin family have been found to be up-regulated in low CO2 conditions, and their expression is controlled by CIA5, a transcription factor that controls many CCM genes. YFP fusions demonstrate that all 3 proteins are located on the thylakoid membrane, and interactome studies indicate that they might associate with chloroplast CCM components. A single mutant defective in BST3 has near-normal growth on low CO2, indicating that the 3 bestrophin-like proteins may have redundant functions. Therefore, an RNA interference (RNAi) approach was adopted to reduce the expression of all 3 genes at once. RNAi mutants with reduced expression of BST1-3 were unable to grow at low CO2 concentrations, exhibited a reduced affinity to inorganic carbon (Ci) compared with the wild-type cells, and showed reduced Ci uptake. We propose that these bestrophin-like proteins are essential components of the CCM that deliver HCO3- accumulated in the chloroplast stroma to CAH3 inside the thylakoid lumen.

Closing the circle.

In Chlamydomonas the different stages of the Calvin-Benson cycle take place in separate locations within the chloroplast.

The many types of carbonic anhydrases in photosynthetic organisms.

Carbonic anhydrases (CAs) are enzymes that catalyze the interconversion of CO2 and HCO3-. In nature, there are multiple families of CA, designated with the Greek letters α through θ. CAs are ubiquitous in plants, algae and photosynthetic bacteria, often playing essential roles in the CO2 concentrating mechanisms (CCMs) which enhance the delivery of CO2 to Rubisco. As algal CCMs become better characterized, it is clear that different types of CAs are playing the same role in different algae. For example, an α-CA catalyzes the conversion of accumulated HCO3- to CO2 in the green alga Chlamydomonas reinhardtii, while a θ-CA performs the same function in the diatom Phaeodactylum tricornutum. In this review we argue that, in addition to its role of delivering CO2 for photosynthesis, other metabolic roles of CA have likely changed as the Earth's atmospheric CO2 level decreased. Since the algal and plant lineages diverged well before the decrease in atmospheric CO2, it is likely that plant, algae and photosynthetic bacteria all adapted independently to the drop in atmospheric CO2. In light of this, we will discuss how the roles of CAs may have changed over time, focusing on the role of CA in pH regulation, how CAs affect CO2 supply for photosynthesis and how CAs may help in the delivery of HCO3- for other metabolic reactions.

Identification and characterization of a solute carrier, CIA8, involved in inorganic carbon acclimation in Chlamydomonas reinhardtii.

The supply of inorganic carbon (Ci) at the site of fixation by Rubisco is a key parameter for efficient CO2 fixation in aquatic organisms including the green alga, Chlamydomonas reinhardtii. Chlamydomonas reinhardtii cells, when grown on limiting CO2, have a CO2-concentrating mechanism (CCM) that functions to concentrate CO2 at the site of Rubisco. Proteins thought to be involved in inorganic carbon uptake have been identified and localized to the plasma membrane or chloroplast envelope. However, current CCM models suggest that additional molecular components are involved in Ci uptake. In this study, the gene Cia8 was identified in an insertional mutagenesis screen and characterized. The protein encoded by Cia8 belongs to the sodium bile acid symporter subfamily. Transcript levels for this gene were significantly up-regulated when the cells were grown on low CO2. The cia8 mutant exhibited reduced growth and reduced affinity for Ci when grown in limiting CO2 conditions. Prediction programs localize this protein to the chloroplast. Ci uptake and the photosynthetic rate, particularly at high external pH, were reduced in the mutant. The results are consistent with the model that CIA8 is involved in Ci uptake in C. reinhardtii.

A robust protocol for efficient generation, and genomic characterization of insertional mutants of Chlamydomonas reinhardtii.

BACKGROUND: Random insertional mutagenesis of Chlamydomonas reinhardtii using drug resistance cassettes has contributed to the generation of tens of thousands of transformants in dozens of labs around the world. In many instances these insertional mutants have helped elucidate the genetic basis of various physiological processes in this model organism. Unfortunately, the insertion sites of many interesting mutants are never defined due to experimental difficulties in establishing the location of the inserted cassette in the Chlamydomonas genome. It is fairly common that several months, or even years of work are conducted with no result. Here we describe a robust method to identify the location of the inserted DNA cassette in the Chlamydomonas genome. RESULTS: Insertional mutants were generated using a DNA cassette that confers paromomycin resistance. This protocol identified the cassette insertion site for greater than 80% of the transformants. In the majority of cases the insertion event was found to be simple, without large deletions of flanking genomic DNA. Multiple insertions were observed in less than 10% of recovered transformants. CONCLUSION: The method is quick, relatively inexpensive and does not require any special equipment beyond an electroporator. The protocol was tailored to ensure that the sequence of the Chlamydomonas genomic DNA flanking the random insertion is consistently obtained in a high proportion of transformants. A detailed protocol is presented to aid in the experimental design and implementation of mutant screens in Chlamydomonas.

Plant Carbonic Anhydrases: Structures, Locations, Evolution, and Physiological Roles.

Carbonic anhydrases (CAs) are zinc metalloenzymes that catalyze the interconversion of CO2 and HCO3- and are ubiquitous in nature. Higher plants contain three evolutionarily distinct CA families, αCAs, ßCAs, and γCAs, where each family is represented by multiple isoforms in all species. Alternative splicing of CA transcripts appears common; consequently, the number of functional CA isoforms in a species may exceed the number of genes. CAs are expressed in numerous plant tissues and in different cellular locations. The most prevalent CAs are those in the chloroplast, cytosol, and mitochondria. This diversity in location is paralleled in the many physiological and biochemical roles that CAs play in plants. In this review, the number and types of CAs in C3, C4, and crassulacean acid metabolism (CAM) plants are considered, and the roles of the α and γCAs are briefly discussed. The remainder of the review focuses on plant ßCAs and includes the identification of homologs between species using phylogenetic approaches, a consideration of the inter- and intracellular localization of the proteins, along with the evidence for alternative splice forms. Current understanding of ßCA tissue-specific expression patterns and what controls them are reviewed, and the physiological roles for which ßCAs have been implicated are presented.

The Cytoplasmic Carbonic Anhydrases ßCA2 and ßCA4 Are Required for Optimal Plant Growth at Low CO2.

Carbonic anhydrases (CAs) are zinc metalloenzymes that interconvert CO2 and HCO3 (-) In plants, both α- and ß-type CAs are present. We hypothesize that cytoplasmic ßCAs are required to modulate inorganic carbon forms needed in leaf cells for carbon-requiring reactions such as photosynthesis and amino acid biosynthesis. In this report, we present evidence that ßCA2 and ßCA4 are the two most abundant cytoplasmic CAs in Arabidopsis (Arabidopsis thaliana) leaves. Previously, ßCA4 was reported to be localized to the plasma membrane, but here, we show that two forms of ßCA4 are expressed in a tissue-specific manner and that the two proteins encoded by ßCA4 localize to two different regions of the cell. Comparing transfer DNA knockout lines with wild-type plants, there was no reduction in the growth rates of the single mutants, ßca2 and ßca4 However, the growth rate of the double mutant, ßca2ßca4, was reduced significantly when grown at 200 µL L(-1) CO2 The reduction in growth of the double mutant was not linked to a reduction in photosynthetic rate. The amino acid content of leaves from the double mutant showed marked reduction in aspartate when compared with the wild type and the single mutants. This suggests the cytoplasmic CAs play an important but not previously appreciated role in amino acid biosynthesis.

CCM8: the eighth international symposium on inorganic carbon uptake by aquatic photosynthetic organisms.

The articles in this special issue of Photosynthesis Research arose from the presentations given at the Eighth International Symposium on Inorganic Carbon Uptake by Aquatic Photosynthetic Organisms held from May 27 to June 1, 2013 in New Orleans, Louisiana USA. The meeting covered all the aspects of CO2 concentrating mechanisms (CCMs) present in photosynthetic bacteria, microalgae and macrophytes, and spanned disciplines from the molecular biology of CCMs to the importance of CCMs in aquatic ecosystems. The publications in this special issue represent our current understanding of CCMs and highlight recent advances in the field. The influences of CCMs on algal biofuel production as well as recent efforts to use the CCM to improve crop plants are also explored.

Carbon allocation and element composition in four Chlamydomonas mutants defective in genes related to the CO2 concentrating mechanism.

Four mutants of Chlamydomonas reinhardtii with defects in different components of the CO2 concentrating mechanism (CCM) or in Rubisco activase were grown autotrophically at high pCO2 and then transferred to low pCO2, in order to study the role of different components of the CCM on carbon allocation and elemental composition. To study carbon allocation, we measured the relative size of the main organic pools by Fourier Transform Infrared spectroscopy. Total reflection X-ray fluorescence was used to analyze the elemental composition of algal cells. Our data show that although the organic pools increased their size at high CO2 in all strains, their stoichiometry was highly homeostatic, i.e., the ratios between carbohydrates and proteins, lipid and proteins, and carbohydrates and lipids, did not change significantly. The only exception was the wild-type 137c, in which proteins decreased relative to carbohydrates and lipids, when the cells were transferred to low CO2. It is noticeable that the two wild types used in this study responded differently to the transition from high to low CO2. Malfunctions of the CCM influenced the concentration of several elements, somewhat altering cell elemental stoichiometry: especially the C/P and N/P ratios changed appreciably in almost all strains as a function of the growth CO2 concentration, except in 137c and the Rubisco activase mutant rca1. In strain cia3, defective in the lumenal carbonic anhydrase (CA), the cell quotas of P, S, Ca, Mn, Fe, and Zn were about 5-fold higher at low CO2 than at high CO2. A Principle Components Analysis showed that, mostly because of its elemental composition, cia3 behaved in a substantially different way from all other strains, at low CO2. The lumenal CA thus plays a crucial role, not only for the correct functioning of the CCM, but also for element utilization. Not surprisingly, growth at high CO2 attenuated differences among strains.