Luke Mackinder

Deformed Wing Virus Implicated in Overwintering Honeybee Colony Losses

ABSTRACT The worldwide decline in honeybee colonies during the past 50 years has often been linked to the spread of the parasitic mite Varroa destructor and its interaction with certain honeybee viruses. Recently in the United States, dramatic honeybee losses (colony collapse disorder) have been reported; however, there remains no clear explanation for these colony losses, with parasitic mites, viruses, bacteria, and fungal diseases all being proposed as possible candidates. Common characteristics that most failing colonies share is a lack of overt disease symptoms and the disappearance of workers from what appears to be normally functioning colonies. In this study, we used quantitative PCR to monitor the presence of three honeybee viruses, deformed wing virus (DWV), acute bee paralysis virus (ABPV), and black queen cell virus (BQCV), during a 1-year period in 15 asymptomatic, varroa mite-positive honeybee colonies in Southern England, and 3 asymptomatic colonies confirmed to be varroa mite free. All colonies with varroa mites underwent control treatments to ensure that mite populations remained low throughout the study. Despite this, multiple virus infections were detected, yet a significant correlation was observed only between DWV viral load and overwintering colony losses. The long-held view has been that DWV is relatively harmless to the overall health status of honeybee colonies unless it is in association with severe varroa mite infestations. Our findings suggest that DWV can potentially act independently of varroa mites to bring about colony losses. Therefore, DWV may be a major factor in overwintering colony losses.

Molecular Mechanisms Underlying Calcification in Coccolithophores

A number of studies are providing increasing genomic and transcriptomic information on the molecular components of transport, and biochemical control in the cell biology of calcification in coccolithophores. In this review we summarise recent evidence for molecular components involved in the trans-cellular transport of Ca2+, inorganic carbon and H+ between the external medium and the intracellular calcification compartment. We present new hypotheses for the transport of substrates to the site of calcification and for the removal of products, highlighting key gaps in our current knowledge. We also discuss how a cellular and molecular approach will improve abilities to understand and predict responses and adaptation to changing ocean chemistry of this important group of microorganisms.

Expression of biomineralization-related ion transport genes in Emiliania huxleyi

Biomineralization in the marine phytoplankton Emiliania huxleyi is a stringently controlled intracellular process. The molecular basis of coccolith production is still relatively unknown although its importance in global biogeochemical cycles and varying sensitivity to increased pCO₂ levels has been well documented. This study looks into the role of several candidate Ca²⁺, H⁺ and inorganic carbon transport genes in E. huxleyi, using quantitative reverse transcriptase PCR. Differential gene expression analysis was investigated in two isogenic pairs of calcifying and non-calcifying strains of E. huxleyi and cultures grown at various Ca²⁺ concentrations to alter calcite production. We show that calcification correlated to the consistent upregulation of a putative HCO₃⁻ transporter belonging to the solute carrier 4 (SLC4) family, a Ca²⁺/H⁺ exchanger belonging to the CAX family of exchangers and a vacuolar H⁺-ATPase. We also show that the coccolith-associated protein, GPA is downregulated in calcifying cells. The data provide strong evidence that these genes play key roles in E. huxleyi biomineralization. Based on the gene expression data and the current literature a working model for biomineralization-related ion transport in coccolithophores is presented.

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.

Actin Is Required for IFT Regulation in Chlamydomonas reinhardtii

Assembly of cilia and flagella requires intraflagellar transport (IFT), a highly regulated kinesin-based transport system that moves cargo from the basal body to the tip of flagella [1]. The recruitment of IFT components to basal bodies is a function of flagellar length, with increased recruitment in rapidly growing short flagella [2]. The molecular pathways regulating IFT are largely a mystery. Because actin network disruption leads to changes in ciliary length and number, actin has been proposed to have a role in ciliary assembly. However, the mechanisms involved are unknown. In Chlamydomonas reinhardtii, conventional actin is found in both the cell body and the inner dynein arm complexes within flagella [3, 4]. Previous work showed that treating Chlamydomonas cells with the actin-depolymerizing compound cytochalasin D resulted in reversible flagellar shortening [5], but how actin is related to flagellar length or assembly remains unknown. Here we utilize small-molecule inhibitors and genetic mutants to analyze the role of actin dynamics in flagellar assembly in Chlamydomonas reinhardtii. We demonstrate that actin plays a role in IFT recruitment to basal bodies during flagellar elongation and that when actin is perturbed, the normal dependence of IFT recruitment on flagellar length is lost. We also find that actin is required for sufficient entry of IFT material into flagella during assembly. These same effects are recapitulated with a myosin inhibitor, suggesting that actin may act via myosin in a pathway by which flagellar assembly is regulated by flagellar length.

Alternative Acetate Production Pathways in Chlamydomonas reinhardtii during Dark Anoxia and the Dominant Role of Chloroplasts in Fermentative Acetate Production

Acetate is a primary Chlamydomonas fermentative product and is linked to dark, anoxic ATP biosynthesis. Chlamydomonas ack/pat mutants were isolated to further characterize fermentation networks, revealing that chloroplast pathways are dominant in this alga, and that despite blocking the primary ATP-generating routes to acetate, Chlamydomonas retains the metabolic flexibility to produce acetate. Chlamydomonas reinhardtii insertion mutants disrupted for genes encoding acetate kinases (EC 2.7.2.1) (ACK1 and ACK2) and a phosphate acetyltransferase (EC 2.3.1.8) (PAT2, but not PAT1) were isolated to characterize fermentative acetate production. ACK1 and PAT2 were localized to chloroplasts, while ACK2 and PAT1 were shown to be in mitochondria. Characterization of the mutants showed that PAT2 and ACK1 activity in chloroplasts plays a dominant role (relative to ACK2 and PAT1 in mitochondria) in producing acetate under dark, anoxic conditions and, surprisingly, also suggested that Chlamydomonas has other pathways that generate acetate in the absence of ACK activity. We identified a number of proteins associated with alternative pathways for acetate production that are encoded on the Chlamydomonas genome. Furthermore, we observed that only modest alterations in the accumulation of fermentative products occurred in the ack1, ack2, and ack1 ack2 mutants, which contrasts with the substantial metabolite alterations described in strains devoid of other key fermentation enzymes.

Critical role of Chlamydomonas reinhardtii ferredoxin-5 in maintaining membrane structure and dark metabolism

Significance Our results suggest that particular ferredoxins in photosynthetic organisms are tailored to serve as electron carriers that sustain day-time and night-time metabolism and that the chloroplast-localized ferredoxin-5 (FDX5) appears to function in the desaturation of fatty acids required for maintaining the correct ratio of the dominant lipids in the thylakoid membranes and the integration of chloroplast and mitochondrial metabolism, which is absolutely required for growth in the dark. The most important messages from this work are that redox components associated with critical activities in photosynthetic organisms must be tuned to the redox conditions of the cells and the overall carbon budget of photosynthetic cells requires an understanding of metabolic features that accompany the movement of cells between light and dark conditions. Photosynthetic microorganisms typically have multiple isoforms of the electron transfer protein ferredoxin, although we know little about their exact functions. Surprisingly, a Chlamydomonas reinhardtii mutant null for the ferredoxin-5 gene (FDX5) completely ceased growth in the dark, with both photosynthetic and respiratory functions severely compromised; growth in the light was unaffected. Thylakoid membranes in dark-maintained fdx5 mutant cells became severely disorganized concomitant with a marked decrease in the ratio of monogalactosyldiacylglycerol to digalactosyldiacylglycerol, major lipids in photosynthetic membranes, and the accumulation of triacylglycerol. Furthermore, FDX5 was shown to physically interact with the fatty acid desaturases CrΔ4FAD and CrFAD6, likely donating electrons for the desaturation of fatty acids that stabilize monogalactosyldiacylglycerol. Our results suggest that in photosynthetic organisms, specific redox reactions sustain dark metabolism, with little impact on daytime growth, likely reflecting the tailoring of electron carriers to unique intracellular metabolic circuits under these two very distinct redox conditions.

A Spatial Interactome Reveals the Protein Organization of the Algal CO2-Concentrating Mechanism

Approximately one-third of global CO2 fixation is performed by eukaryotic algae. Nearly all algae enhance their carbon assimilation by operating a CO2-concentrating mechanism (CCM) built around an organelle called the pyrenoid, whose protein composition is largely unknown. Here, we developed tools in the model alga Chlamydomonas reinhardtii to determine the localizations of 135 candidate CCM proteins and physical interactors of 38 of these proteins. Our data reveal the identity of 89 pyrenoid proteins, including Rubisco-interacting proteins, photosystem I assembly factor candidates, and inorganic carbon flux components. We identify three previously undescribed protein layers of the pyrenoid: a plate-like layer, a mesh layer, and a punctate layer. We find that the carbonic anhydrase CAH6 is in the flagella, not in the stroma that surrounds the pyrenoid as in current models. These results provide an overview of proteins operating in the eukaryotic algal CCM, a key process that drives global carbon fixation.

The Chlamydomonas CO2-concentrating mechanism and its potential for engineering photosynthesis in plants

Contents Summary I. Introduction 54 II. Recent advances in our understanding of the Chlamydomonas CCM 55 III. Current gaps in our understanding of the Chlamydomonas CCM 58 IV. Approaches to rapidly advance our understanding of the Chlamydomonas CCM 58 V. Engineering a CCM into higher plants 58 VI. Conclusion and outlook 59 Acknowledgements 60 References 60 SUMMARY: To meet the food demands of a rising global population, innovative strategies are required to increase crop yields. Improvements in plant photosynthesis by genetic engineering show considerable potential towards this goal. One prospective approach is to introduce a CO2 -concentrating mechanism into crop plants to increase carbon fixation by supplying the central carbon-fixing enzyme, Rubisco, with a higher concentration of its substrate, CO2 . A promising donor organism for the molecular machinery of this mechanism is the eukaryotic alga Chlamydomonas reinhardtii. This review summarizes the recent advances in our understanding of carbon concentration in Chlamydomonas, outlines the most pressing gaps in our knowledge and discusses strategies to transfer a CO2 -concentrating mechanism into higher plants to increase photosynthetic performance.