Chlamydomonas mutants lacking chloroplast TRIOSE PHOSPHATE TRANSPORTER3 are metabolically compromised and light sensitive.

Modulation of photoassimilate export from the chloroplast is essential for controlling the distribution of fixed carbon in the cell and maintaining optimum photosynthetic rates. In this study, we identified chloroplast TRIOSE PHOSPHATE/PHOSPHATE TRANSLOCATOR 2 (CreTPT2) and CreTPT3 in the green alga Chlamydomonas (Chlamydomonas reinhardtii), which exhibit similar substrate specificities but whose encoding genes are differentially expressed over the diurnal cycle. We focused mostly on CreTPT3 because of its high level of expression and the severe phenotype exhibited by tpt3 relative to tpt2 mutants. Null mutants for CreTPT3 had a pleiotropic phenotype that affected growth, photosynthetic activities, metabolite profiles, carbon partitioning, and organelle-specific accumulation of H2O2. These analyses demonstrated that CreTPT3 is a dominant conduit on the chloroplast envelope for the transport of photoassimilates. In addition, CreTPT3 can serve as a safety valve that moves excess reductant out of the chloroplast and appears to be essential for preventing cells from experiencing oxidative stress and accumulating reactive oxygen species, even under low/moderate light intensities. Finally, our studies indicate subfunctionalization of the TRIOSE PHOSPHATE/PHOSPHATE TRANSLOCATOR (CreTPT) transporters and suggest that there are differences in managing the export of photoassimilates from the chloroplasts of Chlamydomonas and vascular plants.

Retrotransposition facilitated the establishment of a primary plastid in the thecate amoeba Paulinella.

The evolution of eukaryotic life was predicated on the development of organelles such as mitochondria and plastids. During this complex process of organellogenesis, the host cell and the engulfed prokaryote became genetically codependent, with the integration of genes from the endosymbiont into the host nuclear genome and subsequent gene loss from the endosymbiont. This process required that horizontally transferred genes become active and properly regulated despite inherent differences in genetic features between donor (endosymbiont) and recipient (host). Although this genetic reorganization is considered critical for early stages of organellogenesis, we have little knowledge about the mechanisms governing this process. The photosynthetic amoeba Paulinella micropora offers a unique opportunity to study early evolutionary events associated with organellogenesis and primary endosymbiosis. This amoeba harbors a "chromatophore," a nascent photosynthetic organelle derived from a relatively recent cyanobacterial association (∼120 million years ago) that is independent of the evolution of primary plastids in plants (initiated ∼1.5 billion years ago). Analysis of the genome and transcriptome of Paulinella revealed that retrotransposition of endosymbiont-derived nuclear genes was critical for their domestication in the host. These retrocopied genes involved in photoprotection in cyanobacteria became expanded gene families and were "rewired," acquiring light-responsive regulatory elements that function in the host. The establishment of host control of endosymbiont-derived genes likely enabled the cell to withstand photo-oxidative stress generated by oxygenic photosynthesis in the nascent organelle. These results provide insights into the genetic mechanisms and evolutionary pressures that facilitated the metabolic integration of the host­endosymbiont association and sustained the evolution of a photosynthetic organelle.

Restricting electron flow at cytochrome b6f when downstream electron acceptors are severely limited.

Photosynthetic organisms frequently experience abiotic stress that restricts their growth and development. Under such circumstances, most absorbed solar energy cannot be used for CO2 fixation and can cause the photoproduction of reactive oxygen species (ROS) that can damage the photosynthetic reaction centers of PSI and PSII, resulting in a decline in primary productivity. This work describes a biological "switch" in the green alga Chlamydomonas reinhardtii that reversibly restricts photosynthetic electron transport (PET) at the cytochrome b6f (Cyt b6f) complex when the capacity for accepting electrons downstream of PSI is severely limited. We specifically show this restriction in STARCHLESS6 (sta6) mutant cells, which cannot synthesize starch when they are limited for nitrogen (growth inhibition) and subjected to a dark-to-light transition. This restriction represents a form of photosynthetic control that causes diminished electron flow to PSI and thereby prevents PSI photodamage but does not appear to rely on a ΔpH. Furthermore, when electron flow is restricted, the plastid alternative oxidase (PTOX) becomes active, functioning as an electron valve that dissipates some excitation energy absorbed by PSII and allows the formation of a proton motive force (PMF) that would drive some ATP production (potentially sustaining PSII repair and nonphotochemical quenching [NPQ]). The restriction at the Cyt b6f complex can be gradually relieved with continued illumination. This study provides insights into how PET responds to a marked reduction in availability of downstream electron acceptors and the protective mechanisms involved.

Chlamydomonas: Fast tracking from genomics.

Elucidating biological processes has relied on the establishment of model organisms, many of which offer advantageous features such as rapid axenic growth, extensive knowledge of their physiological features and gene content, and the ease with which they can be genetically manipulated. The unicellular green alga Chlamydomonas reinhardtii has been an exemplary model that has enabled many scientific breakthroughs over the decades, especially in the fields of photosynthesis, cilia function and biogenesis, and the acclimation of photosynthetic organisms to their environment. Here, we discuss recent molecular/technological advances that have been applied to C. reinhardtii and how they have further fostered its development as a "flagship" algal system. We also explore the future promise of this alga in leveraging advances in the fields of genomics, proteomics, imaging, and synthetic biology for addressing critical future biological issues.

Interplay of four auxiliary factors is required for the assembly of photosystem I reaction center subcomplex.

The photosystem I (PSI) complex consisting of reaction center (RC) subunits, several peripheral subunits and many co-factors, is present in the thylakoid membranes of chloroplasts and cyanobacteria. The assembly of RC subunits (PsaA/B) that bind electron transfer co-factors and antenna pigments is an intricate process, and is mediated by several auxiliary factors such as Ycf3, Y3IP1/CGL59, Ycf4 and Ycf37/PYG7/CGL71. However, their precise molecular mechanisms in RC assembly remain to be addressed. Here we purified four PSI auxiliary factors by affinity chromatography, and characterized co-purified PSI assembly intermediates. We suggest that Ycf3 assists the initial assembly of newly synthesized PsaA/B subunits into an RC subcomplex, while Y3IP1 may be involved in transferring the RC subcomplex from Ycf3 to the Ycf4 module that stabilizes it. CGL71 may form an oligomer that transiently interacts with the PSI RC subcomplex, physically protecting it under oxic conditions until association with the peripheral PSI subunits occurs. Together, our results reveal the interplay among four auxiliary factors required for the stepwise assembly of the PSI RC.

Immunolocalization of Metabolite Transporter Proteins in a Model Cnidarian-Dinoflagellate Symbiosis.

Bidirectional nutrient flow between partners is integral to the cnidarian-dinoflagellate endosymbiosis. However, our current knowledge of the transporter proteins that regulate nutrient and metabolite trafficking is nascent. Four transmembrane transporters that likely play an important role in interpartner nitrogen and carbon exchange were investigated with immunocytochemistry in the model sea anemone Exaiptasia diaphana ("Aiptasia"; strain NZ1): ammonium transporter 1 (AMT1), V-type proton ATPase (VHA), facilitated glucose transporter member 8 (GLUT8), and aquaporin-3 (AQP3). Anemones lacking symbionts were compared with those in symbiosis with either their typical, homologous dinoflagellate symbiont, Breviolum minutum, or the heterologous species, Durusdinium trenchii and Symbiodinium microadriaticum. AMT1 and VHA were only detected in symbiotic Aiptasia, irrespective of symbiont type. However, GLUT8 and AQP3 were detected in both symbiotic and aposymbiotic states. All transporters were localized to both the epidermis and gastrodermis, though localization patterns in host tissues were heavily influenced by symbiont identity, with S. microadriaticum-colonized anemones showing the most distinct patterns. These patterns suggested disruption of fixed carbon and inorganic nitrogen fluxes when in symbiosis with heterologous versus homologous symbionts. This study enhances our understanding of nutrient transport and host-symbiont integration, while providing a platform for further investigation of nutrient transporters and the host-symbiont interface in the cnidarian-dinoflagellate symbiosis. IMPORTANCE Coral reefs are in serious decline, in particular due to the thermally induced dysfunction of the cnidarian-dinoflagellate symbiosis that underlies their success. Yet our ability to react to this crisis is hindered by limited knowledge of how this symbiosis functions. Indeed, we still have much to learn about the cellular integration that determines whether a particular host-symbiont combination can persist, and hence whether corals might be able to adapt by acquiring new, more thermally resistant symbionts. Here, we employed immunocytochemistry to localize and quantify key nutrient transporters in tissues of the sea anemone Aiptasia, a globally adopted model system for this symbiosis, and compared the expression of these transporters when the host is colonized by native versus nonnative symbionts. We showed a clear link between transporter expression and symbiont identity, elucidating the cellular events that dictate symbiosis success, and we provide a methodological platform for further examination of cellular integration in this ecologically important symbiosis.

Symbiosis induces unique volatile profiles in the model cnidarian Aiptasia.

The establishment and maintenance of the symbiosis between a cnidarian host and its dinoflagellate symbionts is central to the success of coral reefs. To explore the metabolite production underlying this symbiosis, we focused on a group of low molecular weight secondary metabolites, biogenic volatile organic compounds (BVOCs). BVOCs are released from an organism or environment, and can be collected in the gas phase, allowing non-invasive analysis of an organism's metabolism (i.e. 'volatilomics'). We characterised volatile profiles of the sea anemone Aiptasia (Exaiptasia diaphana), a model system for cnidarian-dinoflagellate symbiosis, using comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry. We compared volatile profiles between: (1) symbiotic anemones containing their native symbiont, Breviolum minutum; (2) aposymbiotic anemones; and (3) cultured isolates of B. minutum. Overall, 152 BVOCs were detected, and classified into 14 groups based on their chemical structure, the most numerous groups being alkanes and aromatic compounds. A total of 53 BVOCs were differentially abundant between aposymbiotic anemones and B. minutum cultures; 13 between aposymbiotic and symbiotic anemones; and 60 between symbiotic anemones and cultures of B. minutum. More BVOCs were differentially abundant between cultured and symbiotic dinoflagellates than between aposymbiotic and symbiotic anemones, suggesting that symbiosis may modify symbiont physiology more than host physiology. This is the first volatilome analysis of the Aiptasia model system and provides a foundation from which to explore how BVOC production is perturbed under environmental stress, and ultimately the role they play in this important symbiosis.

Alternative outlets for sustaining photosynthetic electron transport during dark-to-light transitions.

Environmental stresses dramatically impact the balance between the production of photosynthetically derived energetic electrons and Calvin-Benson-Bassham cycle (CBBC) activity; an imbalance promotes accumulation of reactive oxygen species and causes cell damage. Hence, photosynthetic organisms have developed several strategies to route electrons toward alternative outlets that allow for storage or harmless dissipation of their energy. In this work, we explore the activities of three essential outlets associated with Chlamydomonas reinhardtii photosynthetic electron transport: (i) reduction of O2 to H2O through flavodiiron proteins (FLVs) and (ii) plastid terminal oxidases (PTOX) and (iii) the synthesis of starch. Real-time measurements of O2 exchange have demonstrated that FLVs immediately engage during dark-to-light transitions, allowing electron transport when the CBBC is not fully activated. Under these conditions, we quantified maximal FLV activity and its overall capacity to direct photosynthetic electrons toward O2 reduction. However, when starch synthesis is compromised, a greater proportion of the electrons is directed toward O2 reduction through both the FLVs and PTOX, suggesting an important role for starch synthesis in priming/regulating CBBC and electron transport. Moreover, partitioning energized electrons between sustainable (starch; energetic electrons are recaptured) and nonsustainable (H2O; energetic electrons are not recaptured) outlets is part of the energy management strategy of photosynthetic organisms that allows them to cope with the fluctuating conditions encountered in nature. Finally, unmasking the repertoire and control of such energetic reactions offers new directions for rational redesign and optimization of photosynthesis to satisfy global demands for food and other resources.

Why is primary endosymbiosis so rare?

Endosymbiosis is a relationship between two organisms wherein one cell resides inside the other. This affiliation, when stable and beneficial for the 'host' cell, can result in massive genetic innovation with the foremost examples being the evolution of eukaryotic organelles, the mitochondria and plastids. Despite its critical evolutionary role, there is limited knowledge about how endosymbiosis is initially established and how host-endosymbiont biology is integrated. Here, we explore this issue, using as our model the rhizarian amoeba Paulinella, which represents an independent case of primary plastid origin that occurred c. 120 million yr ago. We propose the 'chassis and engine' model that provides a theoretical framework for understanding primary plastid endosymbiosis, potentially explaining why it is so rare.

Impact of Menthol on Growth and Photosynthetic Function of Breviolum Minutum (Dinoflagellata, Dinophyceae, Symbiodiniaceae) and Interactions with its Aiptasia Host.

Environmental change, including global warming and chemical pollution, can compromise cnidarian-(e.g., coral-) dinoflagellate symbioses and cause coral bleaching. Understanding the mechanisms that regulate these symbioses will inform strategies for sustaining healthy coral-reef communities. A model system for corals is the symbiosis between the sea anemone Exaiptasia pallida (common name Aiptasia) and its dinoflagellate partners (family Symbiodiniaceae). To complement existing studies of the interactions between these organisms, we examined the impact of menthol, a reagent often used to render cnidarians aposymbiotic, on the dinoflagellate Breviolum minutum, both in culture and in hospite. In both environments, the growth and photosynthesis of this alga were compromised at either 100 or 300 µM menthol. We observed reduction in PSII and PSI functions, the abundances of reaction-center proteins, and, at 300 µM menthol, of total cellular proteins. Interestingly, for free-living algae exposed to 100 µM menthol, an initial decline in growth, photosynthetic activities, pigmentation, and protein abundances reversed after 5-15 d, eventually approaching control levels. This behavior was observed in cells maintained in continuous light, but not in cells experiencing a light-dark regimen, suggesting that B. minutum can detoxify menthol or acclimate and repair damaged photosynthetic complexes in a light- and/or energy-dependent manner. Extended exposures of cultured algae to 300 µM menthol ultimately resulted in algal death. Most symbiotic anemones were also unable to survive this menthol concentration for 30 d. Additionally, cells impaired for photosynthesis by pre-treatment with 300 µM menthol exhibited reduced efficiency in re-populating the anemone host.

The mitochondrial alternative oxidase from Chlamydomonas reinhardtii enables survival in high light.

Photosynthetic organisms often experience extreme light conditions that can cause hyper-reduction of the chloroplast electron transport chain, resulting in oxidative damage. Accumulating evidence suggests that mitochondrial respiration and chloroplast photosynthesis are coupled when cells are absorbing high levels of excitation energy. This coupling helps protect the cells from hyper-reduction of photosynthetic electron carriers and diminishes the production of reactive oxygen species (ROS). To examine this cooperative protection, here we characterized Chlamydomonas reinhardtii mutants lacking the mitochondrial alternative terminal respiratory oxidases, CrAOX1 and CrAOX2. Using fluorescent fusion proteins, we experimentally demonstrated that both enzymes localize to mitochondria. We also observed that the mutant strains were more sensitive than WT cells to high light under mixotrophic and photoautotrophic conditions, with the aox1 strain being more sensitive than aox2 Additionally, the lack of CrAOX1 increased ROS accumulation, especially in very high light, and damaged the photosynthetic machinery, ultimately resulting in cell death. These findings indicate that the Chlamydomonas AOX proteins can participate in acclimation of C. reinhardtii cells to excess absorbed light energy. They suggest that when photosynthetic electron carriers are highly reduced, a chloroplast-mitochondria coupling allows safe dissipation of photosynthetically derived electrons via the reduction of O2 through AOX (especially AOX1)-dependent mitochondrial respiration.

Sub-cellular imaging shows reduced photosynthetic carbon and increased nitrogen assimilation by the non-native endosymbiont Durusdinium trenchii in the model cnidarian Aiptasia.

Hosting different symbiont species can affect inter-partner nutritional fluxes within the cnidarian-dinoflagellate symbiosis. Using nanoscale secondary ion mass spectrometry (NanoSIMS), we measured the spatial incorporation of photosynthetically fixed 13 C and heterotrophically derived 15 N into host and symbiont cells of the model symbiotic cnidarian Aiptasia (Exaiptasia pallida) when colonized with its native symbiont Breviolum minutum or the non-native Durusdinium trenchii. Breviolum minutum exhibited high photosynthetic carbon assimilation per cell and translocation to host tissue throughout symbiosis establishment, whereas D. trenchii assimilated significantly less carbon, but obtained more host nitrogen. These findings suggest that D. trenchii has less potential to provide photosynthetically fixed carbon to the host despite obtaining considerable amounts of heterotrophically derived nitrogen. These sub-cellular events help explain previous observations that demonstrate differential effects of D. trenchii compared to B. minutum on the host transcriptome, proteome, metabolome and host growth and asexual reproduction. Together, these differential effects suggest that the non-native host-symbiont pairing is sub-optimal with respect to the host's nutritional benefits under normal environmental conditions. This contributes to our understanding of the ways in which metabolic integration impacts the benefits of a symbiotic association, and the potential evolution of novel host-symbiont pairings.

Bilin-Dependent Photoacclimation in Chlamydomonas reinhardtii.

In land plants, linear tetrapyrrole (bilin)-based phytochrome photosensors optimize photosynthetic light capture by mediating massive reprogramming of gene expression. But, surprisingly, many green algal genomes lack phytochrome genes. Studies of the heme oxygenase mutant (hmox1) of the green alga Chlamydomonas reinhardtii suggest that bilin biosynthesis in plastids is essential for proper regulation of a nuclear gene network implicated in oxygen detoxification during dark-to-light transitions. hmox1 cannot grow photoautotrophically and photoacclimates poorly to increased illumination. We show that these phenotypes are due to reduced accumulation of photosystem I (PSI) reaction centers, the PSI electron acceptors 5'-monohydroxyphylloquinone and phylloquinone, and the loss of PSI and photosystem II antennae complexes during photoacclimation. The hmox1 mutant resembles chlorophyll biosynthesis mutants phenotypically, but can be rescued by exogenous biliverdin IXα, the bilin produced by HMOX1. This rescue is independent of photosynthesis and is strongly dependent on blue light. RNA-seq comparisons of hmox1, genetically complemented hmox1, and chemically rescued hmox1 reveal that tetrapyrrole biosynthesis and known photoreceptor and photosynthesis-related genes are not impacted in the hmox1 mutant at the transcript level. We propose that a bilin-based, blue-light-sensing system within plastids evolved together with a bilin-based retrograde signaling pathway to ensure that a robust photosynthetic apparatus is sustained in light-grown Chlamydomonas.

Paulinella, a model for understanding plastid primary endosymbiosis.

The uptake and conversion of a free-living cyanobacterium into a photosynthetic organelle by the single-celled Archaeplastida ancestor helped transform the biosphere from low to high oxygen. There are two documented, independent cases of plastid primary endosymbiosis. The first is the well-studied instance in Archaeplastida that occurred ca. 1.6 billion years ago, whereas the second occurred 90-140 million years ago, establishing a permanent photosynthetic compartment (the chromatophore) in amoebae in the genus Paulinella. Here, we briefly summarize knowledge about plastid origin in the Archaeplastida and then focus on Paulinella. In particular, we describe features of the Paulinella chromatophore that make it a model for examining earlier events in the evolution of photosynthetic organelles. Our review stresses recently gained insights into the evolution of chromatophore and nuclear encoded DNA sequences in Paulinella, metabolic connectivity between the endosymbiont and cytoplasm, and systems that target proteins into the chromatophore. We also describe future work with Paulinella, and the potential rewards and challenges associated with developing further this model system.

Optimal nutrient exchange and immune responses operate in partner specificity in the cnidarian-dinoflagellate symbiosis.

The relationship between corals and dinoflagellates of the genus Symbiodinium is fundamental to the functioning of coral ecosystems. It has been suggested that reef corals may adapt to climate change by changing their dominant symbiont type to a more thermally tolerant one, although the capacity for such a shift is potentially hindered by the compatibility of different host-symbiont pairings. Here we combined transcriptomic and metabolomic analyses to characterize the molecular, cellular, and physiological processes that underlie this compatibility, with a particular focus on Symbiodinium trenchii, an opportunistic, thermally tolerant symbiont that flourishes in coral tissues after bleaching events. Symbiont-free individuals of the sea anemone Exaiptasia pallida (commonly referred to as Aiptasia), an established model system for the study of the cnidarian-dinoflagellate symbiosis, were colonized with the "normal" (homologous) symbiont Symbiodinium minutum and the heterologous S. trenchii Analysis of the host gene and metabolite expression profiles revealed that heterologous symbionts induced an expression pattern intermediate between the typical symbiotic state and the aposymbiotic state. Furthermore, integrated pathway analysis revealed that increased catabolism of fixed carbon stores, metabolic signaling, and immune processes occurred in response to the heterologous symbiont type. Our data suggest that both nutritional provisioning and the immune response induced by the foreign "invader" are important factors in determining the capacity of corals to adapt to climate change through the establishment of novel symbioses.

Insights into the red algae and eukaryotic evolution from the genome of Porphyra umbilicalis (Bangiophyceae, Rhodophyta).

Porphyra umbilicalis (laver) belongs to an ancient group of red algae (Bangiophyceae), is harvested for human food, and thrives in the harsh conditions of the upper intertidal zone. Here we present the 87.7-Mbp haploid Porphyra genome (65.8% G + C content, 13,125 gene loci) and elucidate traits that inform our understanding of the biology of red algae as one of the few multicellular eukaryotic lineages. Novel features of the Porphyra genome shared by other red algae relate to the cytoskeleton, calcium signaling, the cell cycle, and stress-tolerance mechanisms including photoprotection. Cytoskeletal motor proteins in Porphyra are restricted to a small set of kinesins that appear to be the only universal cytoskeletal motors within the red algae. Dynein motors are absent, and most red algae, including Porphyra, lack myosin. This surprisingly minimal cytoskeleton offers a potential explanation for why red algal cells and multicellular structures are more limited in size than in most multicellular lineages. Additional discoveries further relating to the stress tolerance of bangiophytes include ancestral enzymes for sulfation of the hydrophilic galactan-rich cell wall, evidence for mannan synthesis that originated before the divergence of green and red algae, and a high capacity for nutrient uptake. Our analyses provide a comprehensive understanding of the red algae, which are both commercially important and have played a major role in the evolution of other algal groups through secondary endosymbioses.

A giant type I polyketide synthase participates in zygospore maturation in Chlamydomonas reinhardtii.

Polyketide synthases (PKSs) occur in many bacteria, fungi and plants. They are highly versatile enzymes involved in the biosynthesis of a large variety of compounds including antimicrobial agents, polymers associated with bacterial cell walls and plant pigments. While harmful algae are known to produce polyketide toxins, sequences of the genomes of non-toxic algae, including those of many green algal species, have surprisingly revealed the presence of genes encoding type I PKSs. The genome of the model alga Chlamydomonas reinhardtii (Chlorophyta) contains a single type I PKS gene, designated PKS1 (Cre10.g449750), which encodes a giant PKS with a predicted mass of 2.3 MDa. Here, we show that PKS1 is induced in 2-day-old zygotes and is required for their development into zygospores, the dormant stage of the zygote. Wild-type zygospores contain knob-like structures (~50 nm diameter) that form at the cell surface and develop a central cell wall layer; both of these structures are absent from homozygous pks1 mutants. Additionally, in contrast to wild-type zygotes, chlorophyll degradation is delayed in homozygous pks1 mutant zygotes, indicating a disruption in zygospore development. In agreement with the role of the PKS in the formation of the highly resistant zygospore wall, mutant zygotes have lost the formidable desiccation tolerance of wild-type zygotes. Together, our results represent functional analyses of a PKS mutant in a photosynthetic eukaryotic microorganism, revealing a central function for polyketides in the sexual cycle and survival under stressful environmental conditions.

GreenCut protein CPLD49 of Chlamydomonas reinhardtii associates with thylakoid membranes and is required for cytochrome b6 f complex accumulation.

The GreenCut encompasses a suite of nucleus-encoded proteins with orthologs among green lineage organisms (plants, green algae), but that are absent or poorly conserved in non-photosynthetic/heterotrophic organisms. In Chlamydomonas reinhardtii, CPLD49 (Conserved in Plant Lineage and Diatoms49) is an uncharacterized GreenCut protein that is critical for maintaining normal photosynthetic function. We demonstrate that a cpld49 mutant has impaired photoautotrophic growth under high-light conditions. The mutant exhibits a nearly 90% reduction in the level of the cytochrome b6 f complex (Cytb6 f), which impacts linear and cyclic electron transport, but does not compromise the ability of the strain to perform state transitions. Furthermore, CPLD49 strongly associates with thylakoid membranes where it may be part of a membrane protein complex with another GreenCut protein, CPLD38; a mutant null for CPLD38 also impacts Cytb6 f complex accumulation. We investigated several potential functions of CPLD49, with some suggested by protein homology. Our findings are congruent with the hypothesis that CPLD38 and CPLD49 are part of a novel thylakoid membrane complex that primarily modulates accumulation, but also impacts the activity of the Cytb6 f complex. Based on motifs of CPLD49 and the activities of other CPLD49-like proteins, we suggest a role for this putative dehydrogenase in the synthesis of a lipophilic thylakoid membrane molecule or cofactor that influences the assembly and activity of Cytb6 f.

Glucose-Induced Trophic Shift in an Endosymbiont Dinoflagellate with Physiological and Molecular Consequences.

Interactions between the dinoflagellate endosymbiont Symbiodinium and its cnidarian hosts (e.g. corals, sea anemones) are the foundation of coral-reef ecosystems. Carbon flow between the partners is a hallmark of this mutualism, but the mechanisms governing this flow and its impact on symbiosis remain poorly understood. We showed previously that although Symbiodinium strain SSB01 can grow photoautotrophically, it can grow mixotrophically or heterotrophically when supplied with Glc, a metabolite normally transferred from the alga to its host. Here we show that Glc supplementation of SSB01 cultures causes a loss of pigmentation and photosynthetic activity, disorganization of thylakoid membranes, accumulation of lipid bodies, and alterations of cell-surface morphology. We used global transcriptome analyses to determine if these physiological changes were correlated with changes in gene expression. Glc-supplemented cells exhibited a marked reduction in levels of plastid transcripts encoding photosynthetic proteins, although most nuclear-encoded transcripts (including those for proteins involved in lipid synthesis and formation of the extracellular matrix) exhibited little change in their abundances. However, the altered carbon metabolism in Glc-supplemented cells was correlated with modest alterations (approximately 2x) in the levels of some nuclear-encoded transcripts for sugar transporters. Finally, Glc-bleached SSB01 cells appeared unable to efficiently populate anemone larvae. Together, these results suggest links between energy metabolism and cellular physiology, morphology, and symbiotic interactions. However, the results also show that in contrast to many other organisms, Symbiodinium can undergo dramatic physiological changes that are not reflected by major changes in the abundances of nuclear-encoded transcripts and thus presumably reflect posttranscriptional regulatory processes.

An Indexed, Mapped Mutant Library Enables Reverse Genetics Studies of Biological Processes in Chlamydomonas reinhardtii.

The green alga Chlamydomonas reinhardtii is a leading unicellular model for dissecting biological processes in photosynthetic eukaryotes. However, its usefulness has been limited by difficulties in obtaining mutants in specific genes of interest. To allow generation of large numbers of mapped mutants, we developed high-throughput methods that (1) enable easy maintenance of tens of thousands of Chlamydomonas strains by propagation on agar media and by cryogenic storage, (2) identify mutagenic insertion sites and physical coordinates in these collections, and (3) validate the insertion sites in pools of mutants by obtaining >500 bp of flanking genomic sequences. We used these approaches to construct a stably maintained library of 1935 mapped mutants, representing disruptions in 1562 genes. We further characterized randomly selected mutants and found that 33 out of 44 insertion sites (75%) could be confirmed by PCR, and 17 out of 23 mutants (74%) contained a single insertion. To demonstrate the power of this library for elucidating biological processes, we analyzed the lipid content of mutants disrupted in genes encoding proteins of the algal lipid droplet proteome. This study revealed a central role of the long-chain acyl-CoA synthetase LCS2 in the production of triacylglycerol from de novo-synthesized fatty acids.