Paulinella chromatophora is a cercozoan amoeba that contains "chromatophores," which are photosynthetic inclusions of cyanobacterial origin. The recent discovery that chromatophores evolved independently of plastids, underwent major genome reduction, and transferred at least two genes to the host nucleus has highlighted P. chromatophora as a model to infer early steps in the evolution of photosynthetic organelles. However, owing to the paucity of nuclear genome sequence data, the extent of endosymbiotic gene transfer (EGT) and host symbiont regulation are currently unknown. A combination of 454 and Illumina next generation sequencing enabled us to generate a comprehensive reference transcriptome data set for P. chromatophora on which we mapped short Illumina cDNA reads generated from cultures from the dark and light phases of a diel cycle. Combined with extensive phylogenetic analyses of the deduced protein sequences, these data revealed that 1) about 0.3-0.8% of the nuclear genes were obtained by EGT compared with 11-14% in the Plantae, 2) transferred genes show a distinct bias in that many encode small proteins involved in photosynthesis and photoacclimation, 3) host cells established control over expression of transferred genes, and 4) not only EGT, but to a minor extent also horizontal gene transfer from organisms that presumably served as food sources, helped to shape the nuclear genome of P. chromatophora. The identification of a significant number of transferred genes involved in photosynthesis and photoacclimation of thylakoid membranes as well as the observed transcriptional regulation of these genes strongly implies import of the encoded gene products into chromatophores, a feature previously thought to be restricted to canonical organelles. Thus, a possible mechanism by which P. chromatophora exerts control over the performance of its newly acquired photosynthetic organelle may involve controlling the expression of nuclear-encoded chromatophore-targeted regulatory components of the thylakoid membranes.

A sensor histidine kinase of Synechococcus sp. strain PCC7942, designated nblS, was previously identified and shown to be critical for the acclimation of cells to high-light and nutrient limitation conditions and to influence the expression of a number of light-responsive genes. The nblS orthologue in Synechocystis sp. strain PCC6803 is designated dspA (also called hik33). We have generated a dspA null mutant and analyzed global gene expression in both the mutant and wild-type strains under high- and low-light conditions. The mutant is aberrant for the expression of many genes encoding proteins critical for photosynthesis, phosphate and carbon acquisition, and the amelioration of stress conditions. Furthermore, transcripts from a number of genes normally detected only during exposure of wild-type cells to high-light conditions become partially constitutive in the low-light-grown dspA mutant. Other genes for which transcripts decline upon exposure of wild-type cells to high light are already lower in the mutant during growth in low light. These results suggest that DspA may influence gene expression in both a positive and a negative manner and that the dspA mutant behaves as if it were experiencing stress conditions (e.g., high-light exposure) even when maintained at near-optimal growth conditions for wild-type cells. This is discussed with respect to the importance of DspA for regulating the responses of the cell to environmental cues.

The Chlamydomonas reinhardtii transcription factor PSR1 is required for the control of activities involved in scavenging phosphate from the environment during periods of phosphorus limitation. Increased scavenging activity reflects the development of high-affinity phosphate transport and the expression of extracellular phosphatases that can cleave phosphate from organic compounds in the environment. A comparison of gene expression patterns using microarray analyses and quantitative PCRs with wild-type and psr1 mutant cells deprived of phosphorus has revealed that PSR1 also controls genes encoding proteins with potential "electron valve" functions-these proteins can serve as alternative electron acceptors that help prevent photodamage caused by overexcitation of the photosynthetic electron transport system. In accordance with this finding, phosphorus-starved psr1 mutants die when subjected to elevated light intensities; at these intensities, the wild-type cells still exhibit rapid growth. Acclimation to phosphorus deprivation also involves a reduction in the levels of transcripts encoding proteins involved in photosynthesis and both cytoplasmic and chloroplast translation as well as an increase in the levels of transcripts encoding stress-associated chaperones and proteases. Surprisingly, phosphorus-deficient psr1 cells (but not wild-type cells) also display expression patterns associated with specific responses to sulfur deprivation, suggesting a hitherto unsuspected link between the signal transduction pathways involved in controlling phosphorus and sulfur starvation responses. Together, these results demonstrate that PSR1 is critical for the survival of cells under conditions of suboptimal phosphorus availability and that it plays a key role in controlling both scavenging responses and the ability of the cell to manage excess absorbed excitation energy.

Global photosynthesis consumes ten times more CO2 than net anthropogenic emissions, and microalgae account for nearly half of this consumption(1). The high efficiency of algal photosynthesis relies on a mechanism concentrating CO2 (CCM) at the catalytic site of the carboxylating enzyme RuBisCO, which enhances CO2 fixation(2). Although many cellular components involved in the transport and sequestration of inorganic carbon have been identified(3,4), how microalgae supply energy to concentrate CO2 against a thermodynamic gradient remains unknown(4-6). Here we show that in the green alga Chlamydomonas reinhardtii, the combined action of cyclic electron flow and O-2 photoreduction-which depend on PGRL1 and flavodiiron proteins, respectively-generate a low luminal pH that is essential for CCM function. We suggest that luminal protons are used downstream of thylakoid bestrophin-like transporters, probably for the conversion of bicarbonate to CO2. We further establish that an electron flow from chloroplast to mitochondria contributes to energizing non-thylakoid inorganic carbon transporters, probably by supplying ATP. We propose an integrated view of the network supplying energy to the CCM, and describe how algal cells distribute energy from photosynthesis to power different CCM processes. These results suggest a route for the transfer of a functional algal CCM to plants to improve crop productivity.

Responses of photosynthetic organisms to sulfur starvation include (i) increasing the capacity of the cell for transporting and/or assimilating exogenous sulfate, (ii) restructuring cellular features to conserve sulfur resources, and (iii) modulating metabolic processes and rates of cell growth and division. We used microarray analyses to obtain a genome-level view of changes in mRNA abundances in the green alga Chlamydomonas reinhardtii during sulfur starvation. The work confirms and extends upon previous findings showing that sulfur deprivation elicits changes in levels of transcripts for proteins that help scavenge sulfate and economize on the use of sulfur resources. Changes in levels of transcripts encoding members of the light-harvesting polypeptide family, such as LhcSR2, suggest restructuring of the photosynthetic apparatus during sulfur deprivation. There are also significant changes in levels of transcripts encoding enzymes involved in metabolic processes (e.g., carbon metabolism), intracellular proteolysis, and the amelioration of oxidative damage; a marked and sustained increase in mRNAs for a putative vanadium chloroperoxidase and a peroxiredoxin may help prolong survival of C. reinhardtii during sulfur deprivation. Furthermore, many of the sulfur stress-regulated transcripts (encoding polypeptides associated with sulfate uptake and assimilation, oxidative stress, and photosynthetic function) are not properly regulated in the sac1 mutant of C. reinhardtii, a strain that dies much more rapidly than parental cells during sulfur deprivation. Interestingly, sulfur stress elicits dramatic changes in levels of transcripts encoding putative chloroplast-localized chaperones in the sac1 mutant but not in the parental strain. These results suggest various strategies used by photosynthetic organisms during acclimation to nutrient-limited growth.

Photosynthetic organisms use sunlight as the primary energy source to fix CO2. However, in nature, light energy is highly variable, reaching levels of saturation for periods ranging from milliseconds to hours. In the green microalga Chlamydomonas reinhardtii, safe dissipation of excess light energy by non-photochemical quenching (NPQ) is mediated by light-harvesting complex stress-related (LHCSR) proteins and redistribution of light-harvesting antennae between the photosystems (state transition). Although each component underlying NPQ has been documented, their relative contributions to NPQ under fluctuating light conditions remain unknown. Here, by monitoring NPQ in intact cells throughout high light/dark cycles of various illumination periods, we find that the dynamics of NPQ depend on the timescales of light fluctuations. We show that LHCSRs play a major role during the light phases of light fluctuations and describe their role in growth under rapid light fluctuations. We further reveal an activation of NPQ during the dark phases of all high light/dark cycles and show that this phenomenon arises from state transition. Finally, we show that LHCSRs and state transition synergistically cooperate to enable NPQ response during light fluctuations. These results highlight the dynamic functioning of photoprotection under light fluctuations and open a new way to systematically characterize the photosynthetic response to an ever-changing light environment.

When exposed to stress such as high seawater temperature, corals and other cnidarians can bleach due to loss of symbiotic algae from the host tissue and/or loss of pigments from the algae. Although the environmental conditions that trigger bleaching are reasonably well known, its cellular and molecular mechanisms are not well understood. Previous studies have reported the occurrence of at least four different cellular mechanisms for the loss of symbiotic algae from the host tissue: in situ degradation of algae, exocytic release of algae from the host, detachment of host cells containing algae, and death of host cells containing algae. The relative contributions of these several mechanisms to bleaching remain unclear, and it is also not known whether these relative contributions change in animals subjected to different types and/or durations of stresses. In this study, we used a clonal population of the small sea anemone Aiptasia, exposed individuals to various precisely controlled stress conditions, and quantitatively assessed the several possible bleaching mechanisms in parallel. Under all stress conditions tested, except for acute cold shock at 4 degrees C, expulsion of intact algae from the host cells appeared to be by far the predominant mechanism of bleaching. During acute cold shock, in situ degradation of algae and host-cell detachment also became quantitatively significant, and the algae released under these conditions appeared to be severely damaged.

Coral reef ecosystems are metabolically founded on the mutualism between corals and photosynthetic dinoflagellates of the genus Symbiodinium. The glass anemone Aiptasia sp. has become a tractable model for this symbiosis, and recent advances in genetic information have enabled the use of mass spectrometry-based proteomics in this model. We utilized label-free liquid chromatography electrospray-ionization tandem mass spectrometry to analyze the effects of symbiosis on the proteomes of symbiotic and aposymbiotic Aiptasia. We identified and obtained relative quantification of more than 3,300 proteins in 1,578 protein clusters, with 81 protein clusters showing significantly different expression between symbiotic states. Symbiotic anemones showed significantly higher expression of proteins involved in lipid storage and transport, nitrogen transport and cycling, intracellular trafficking, endocytosis and inorganic carbon transport. These changes reflect shifts in host metabolism and nutrient reserves due to increased nutritional exchange with the symbionts, as well as mechanisms for supplying inorganic nutrients to the algae. Aposymbiotic anemones exhibited increased expression of multiple systems responsible for mediating reactive oxygen stress, suggesting that the host derives direct or indirect protection from oxidative stress while in symbiosis. Aposymbiotic anemones also increased their expression of an array of proteases and chitinases, indicating a metabolic shift from autotrophy to heterotrophy. These results provide a comprehensive Aiptasia proteome with more direct relative quantification of protein abundance than transcriptomic methods. The extension of omics techniques to this model system will allow more powerful studies of coral physiology, ecosystem function, and the effects of biotic and abiotic stress on the coral-dinoflagellate mutualism.

Plastids, the photosynthetic organelles, originated >1 billion y ago via the endosymbiosis of a cyanobacterium. The resulting proliferation of primary producers fundamentally changed global ecology. Endosymbiotic gene transfer (EGT) from the intracellular cyanobacterium to the nucleus is widely recognized as a critical factor in the evolution of photosynthetic eukaryotes. The contribution of horizontal gene transfers (HGTs) from other bacteria to plastid establishment remains more controversial. A novel perspective on this issue is provided by the amoeba Paulinella chromatophora, which contains photosynthetic organelles (chromatophores) that are only 60-200 million years old. Chromatophore genome reduction entailed the loss of many biosynthetic pathways including those for numerous amino acids and cofactors. How the host cell compensates for these losses remains unknown, because the presence of bacteria in all available P. chromatophora cultures excluded elucidation of the full metabolic capacity and occurrence of HGT in this species. Here we generated a high-quality transcriptome and draft genome assembly from the first bacteria-free P. chromatophora culture to deduce rules that govern organelle integration into cellular metabolism. Our analyses revealed that nuclear and chromatophore gene inventories provide highly complementary functions. At least 229 nuclear genes were acquired via HGT from various bacteria, of which only 25% putatively arose through EGT from the chromatophore genome. Many HGT-derived bacterial genes encode proteins that fill gaps in critical chromatophore pathways/processes. Our results demonstrate a dominant role for HGT in compensating for organelle genome reduction and suggest that phagotrophy may be a major driver of HGT.

Background: Bacterial endosymbionts are found across the eukaryotic kingdom and profoundly impacted eukaryote evolution. In many endosymbiotic associations with vertically inherited symbionts, highly complementary metabolic functions encoded by host and endosymbiont genomes indicate integration of metabolic processes between the partner organisms. While endosymbionts were initially expected to exchange only metabolites with their hosts, recent evidence has demonstrated that also host-encoded proteins can be targeted to the bacterial symbionts in various endosymbiotic systems. These proteins seem to participate in regulating symbiont growth and physiology. However, mechanisms required for protein targeting and the specific endosymbiont targets of these trafficked proteins are currently unexplored owing to a lack of molecular tools that enable functional studies of endosymbiotic systems.