Moritz Meyer

Functional hybrid rubisco enzymes with plant small subunits and algal large subunits: engineered rbcS cDNA for expression in chlamydomonas.

There has been much interest in the chloroplast-encoded large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) as a target for engineering an increase in net CO2 fixation in photosynthesis. Improvements in the enzyme would lead to an increase in the production of food, fiber, and renewable energy. Although the large subunit contains the active site, a family of rbcS nuclear genes encodes the Rubisco small subunits, which can also influence the carboxylation catalytic efficiency and CO2/O2 specificity of the enzyme. To further define the role of the small subunit in Rubisco function, small subunits from spinach, Arabidopsis, and sunflower were assembled with algal large subunits by transformation of a Chlamydomonas reinhardtii mutant that lacks the rbcS gene family. Foreign rbcS cDNAs were successfully expressed in Chlamydomonas by fusing them to a Chlamydomonas rbcS transit peptide sequence engineered to contain rbcS introns. Although plant Rubisco generally has greater CO2/O2 specificity but a lower carboxylation Vmax than Chlamydomonas Rubisco, the hybrid enzymes have 3–11% increases in CO2/O2 specificity and retain near normal Vmax values. Thus, small subunits may make a significant contribution to the overall catalytic performance of Rubisco. Despite having normal amounts of catalytically proficient Rubisco, the hybrid mutant strains display reduced levels of photosynthetic growth and lack chloroplast pyrenoids. It appears that small subunits contain the structural elements responsible for targeting Rubisco to the algal pyrenoid, which is the site where CO2 is concentrated for optimal photosynthesis.

Origins and diversity of eukaryotic CO2-concentrating mechanisms: lessons for the future

The importance of the eukaryotic algal CO(2)-concentrating mechanism (CCM) is considered in terms of global productivity as well as molecular phylogeny and diversity. The three major constituents comprising the CCM in the majority of eukaryotes are described. These include: (i) likely plasma- and chloroplast-membrane inorganic carbon transporters; (ii) a suite of carbonic anhydrase enzymes in strategic locations; and usually (iii) a microcompartment in which most Rubisco aggregates (the chloroplast pyrenoid). The molecular diversity of known CCM components are set against the current green algal model for their probable operation. The review then focuses on the kinetic and cystallographic interactions of Rubisco, which permit pyrenoid formation and CCM function. Firstly, we consider observations that surface residues of the Rubisco small subunit directly condition Rubisco aggregation and pyrenoid formation. Secondly, we reanalyse the phylogenetic progression in green Rubisco kinetic properties, and suggest that Rubisco substrate selectivity (the specificity factor, S(rel), and affinity for CO(2), K(c)) demonstrate a systematic relaxation, which directly relates to the origins and effectiveness of a CCM. Finally, we consider the implications of eukaryotic CCM regulation and minimum components needed for introduction into higher plants as a possible means to enhance crop productivity in the future.

Rubisco small-subunit α-helices control pyrenoid formation in Chlamydomonas

The pyrenoid is a subcellular microcompartment in which algae sequester the primary carboxylase, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The pyrenoid is associated with a CO2-concentrating mechanism (CCM), which improves the operating efficiency of carbon assimilation and overcomes diffusive limitations in aquatic photosynthesis. Using the model alga Chlamydomonas reinhardtii, we show that pyrenoid formation, Rubisco aggregation, and CCM activity relate to discrete regions of the Rubisco small subunit (SSU). Specifically, pyrenoid occurrence was shown to be conditioned by the amino acid composition of two surface-exposed α-helices of the SSU: higher plant-like helices knock out the pyrenoid, whereas native algal helices establish a pyrenoid. We have also established that pyrenoid integrity was essential for the operation of an active CCM. With the algal CCM being functionally analogous to the terrestrial C4 pathway in higher plants, such insights may offer a route toward transforming algal and higher plant productivity for the future.

To concentrate or ventilate? Carbon acquisition, isotope discrimination and physiological ecology of early land plant life forms

A comparative study has been made of the photosynthetic physiological ecology and carbon isotope discrimination characteristics for modern-day bryophytes and closely related algal groups. Firstly, the extent of bryophyte distribution and diversification as compared with more advanced land plant groups is considered. Secondly, measurements of instantaneous carbon isotope discrimination (Δ), photosynthetic CO2 assimilation and electron transport rates were compared during the drying cycles. The extent of surface diffusion limitation (when wetted), internal conductance and water use efficiency (WUE) at optimal tissue water content (TWC) were derived for liverworts and a hornwort from contrasting habitats and with differing degrees of thallus ventilation (as intra-thalline cavities and internal airspaces). We also explore how the operation of a biophysical carbon-concentrating mechanism (CCM) tempers isotope discrimination characteristics in two other hornworts, as well as the green algae Coleochaete orbicularis and Chlamydomonas reinhardtii. The magnitude of Δ was compared for each life form over a drying curve and used to derive the surface liquid-phase conductance (when wetted) and internal conductance (at optimal TWC). The magnitude of external and internal conductances, and WUE, was higher for ventilated, compared with non-ventilated, liverworts and hornworts, but the values were similar within each group, suggesting that both factors have been optimized for each life form. For the hornworts, leakiness of the CCM was highest for Megaceros vincentianus and C. orbicularis (approx. 30%) and, at 5%, lowest in C. reinhardtii grown under ambient CO2 concentrations. Finally, evidence for the operation of a CCM in algae and hornworts is considered in terms of the probable role of the chloroplast pyrenoid, as the origins, structure and function of this enigmatic organelle are explored during the evolution of land plants.

A repeat protein links Rubisco to form the eukaryotic carbon-concentrating organelle

Significance Eukaryotic algae, which play a fundamental role in global CO2 fixation, enhance the performance of the carbon-fixing enzyme Rubisco by placing it into an organelle called the pyrenoid. Despite the ubiquitous presence and biogeochemical importance of this organelle, how Rubisco assembles to form the pyrenoid remains a long-standing mystery. Our discovery of an abundant repeat protein that binds Rubisco in the pyrenoid represents a critical advance in our understanding of pyrenoid biogenesis. The repeat sequence of this protein suggests elegant models to explain the structural arrangement of Rubisco enzymes in the pyrenoid. Beyond advances in basic understanding, our findings open doors to the engineering of algal pyrenoids into crops to enhance yields. Biological carbon fixation is a key step in the global carbon cycle that regulates the atmospheres composition while producing the food we eat and the fuels we burn. Approximately one-third of global carbon fixation occurs in an overlooked algal organelle called the pyrenoid. The pyrenoid contains the CO2-fixing enzyme Rubisco and enhances carbon fixation by supplying Rubisco with a high concentration of CO2. Since the discovery of the pyrenoid more that 130 y ago, the molecular structure and biogenesis of this ecologically fundamental organelle have remained enigmatic. Here we use the model green alga Chlamydomonas reinhardtii to discover that a low-complexity repeat protein, Essential Pyrenoid Component 1 (EPYC1), links Rubisco to form the pyrenoid. We find that EPYC1 is of comparable abundance to Rubisco and colocalizes with Rubisco throughout the pyrenoid. We show that EPYC1 is essential for normal pyrenoid size, number, morphology, Rubisco content, and efficient carbon fixation at low CO2. We explain the central role of EPYC1 in pyrenoid biogenesis by the finding that EPYC1 binds Rubisco to form the pyrenoid matrix. We propose two models in which EPYC1’s four repeats could produce the observed lattice arrangement of Rubisco in the Chlamydomonas pyrenoid. Our results suggest a surprisingly simple molecular mechanism for how Rubisco can be packaged to form the pyrenoid matrix, potentially explaining how Rubisco packaging into a pyrenoid could have evolved across a broad range of photosynthetic eukaryotes through convergent evolution. In addition, our findings represent a key step toward engineering a pyrenoid into crops to enhance their carbon fixation efficiency.

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

Summary Many eukaryotic green algae possess biophysical carbon‐concentrating mechanisms (CCMs) that enhance photosynthetic efficiency and thus permit high growth rates at low CO 2 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 H14 CO 3 ‐ 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.

Dynamics of Carbon-Concentrating Mechanism Induction and Protein Relocalization during the Dark-to-Light Transition in Synchronized Chlamydomonas reinhardtii

In synchronized Chlamydomonas reinhardtii cells, the carbon-concentrating mechanism is induced prior to dawn, which coincides with the relocalization of key proteins to the chloroplast pyrenoid. 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.

Will an algal CO2-concentrating mechanism work in higher plants?

Many algae use a biophysical carbon concentrating mechanism for active accumulation and retention of inorganic carbon within chloroplasts, with CO2 fixation by RuBisCO within a micro-compartment, the pyrenoid. Engineering such mechanisms into higher plant chloroplasts is a possible route to augment RuBisCO operating efficiency and photosynthetic rates. Significant progress has been made recently in characterising key algal transporters and identifying factors responsible for the aggregation of RuBisCO into the pyrenoid. Several transporters have now also been successfully incorporated into higher plant chloroplasts. Consistent with the predictions from modelling, regulation of higher plant plastidic carbonic anhydrases and some form of RuBisCO aggregation will be needed before the mechanism delivers potential benefits. Key research priorities include a better understanding of the regulation of the algal carbon concentrating mechanism, advancing the fundamental characterisation of known components, evaluating whether higher plant chloroplasts can accommodate a pyrenoid, and, ultimately, testing transgenic lines under realistic growth conditions.

Rubisco small subunits from the unicellular green alga Chlamydomonas complement Rubisco-deficient mutants of Arabidopsis

Summary Introducing components of algal carbon concentrating mechanisms (CCMs) into higher plant chloroplasts could increase photosynthetic productivity. A key component is the Rubisco‐containing pyrenoid that is needed to minimise CO 2 retro‐diffusion for CCM operating efficiency. Rubisco in Arabidopsis was re‐engineered to incorporate sequence elements that are thought to be essential for recruitment of Rubisco to the pyrenoid, namely the algal Rubisco small subunit (SSU, encoded by rbcS) or only the surface‐exposed algal SSU α‐helices. Leaves of Arabidopsis rbcs mutants expressing ‘pyrenoid‐competent’ chimeric Arabidopsis SSUs containing the SSU α‐helices from Chlamydomonas reinhardtii can form hybrid Rubisco complexes with catalytic properties similar to those of native Rubisco, suggesting that the α‐helices are catalytically neutral. The growth and photosynthetic performance of complemented Arabidopsis rbcs mutants producing near wild‐type levels of the hybrid Rubisco were similar to those of wild‐type controls. Arabidopsis rbcs mutants expressing a Chlamydomonas SSU differed from wild‐type plants with respect to Rubisco catalysis, photosynthesis and growth. This confirms a role for the SSU in influencing Rubisco catalytic properties.

Pyrenoid loss in Chlamydomonas reinhardtii causes limitations in CO2 supply, but not thylakoid operating efficiency

The Chlamydomonas reinhardtii pyrenoid is a Rubisco-containing microcompartment primarily responsible for facilitating carbon accumulation and does not affect thylakoid membrane photosynthetic energetics.