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Under ideal conditions, Chlamydomonas reinhardtii can photoacclimate to excess light through various short- and long-term mechanisms. However, how microalgae handle excess light stress once they exit exponential growth, and especially in stationary phase, is less understood. Our study explored C. reinhardtii's photoprotection capacity and acclimation strategies during high-light stress once batch culture growth reached stationary phase. We monitored cultures of wildtype strain (CC125) over five days once they reached stationary phase under both low-light (LL) and high-light (HL) conditions. Under HL, many photosynthetic proteins were degraded but the stress-related light harvesting complex protein (LHCSR) was rapidly induced and contributed to the rapid activation of nonphotochemical quenching (NPQ). However, the LHCSR3-defective mutant (CC4614, npq4) lacked the rapid induction of quenching typical of post-exponential cultures, indicating that LHCSR3 is required for this response in stationary phase. Collectively, the main strategy for photoacclimation in stationary phase appears to be a dramatic reduction of photosystems while maintaining LHCII-LHCSR antenna complexes that prime the antenna for rapid activation of quenching upon light exposure. Part of this response to HL involves a resumption of cell growth after two days, that we hypothesized is due to the stimulation of growth-regulating pathways due to increased metabolite pools from the HL-induced protein turnover in the cell, something that remains to be tested. These findings demonstrate how C. reinhardtii manages high-light stress during stationary phases to maximize longevity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Although life-history trade-offs are central to life-history evolution, their mechanistic basis is often unclear. Traditionally, trade-offs are understood in terms of competition for limited resources among traits within an organism, which could be mediated by signal transduction pathways at the level of cellular metabolism. Nevertheless, trade-offs are also thought to be produced as a consequence of the performance of one activity generating negative consequences for other traits, or the result of genes or pathways that simultaneously regulate two life-history traits in opposite directions (antagonistic pleiotropy), independent of resource allocation. Yet examples of genes with antagonistic effects on life-history traits are limited. This study provides direct evidence for a gene- RLS1 , that is involved in increasing survival in nutrient-limiting environments at a cost to immediate reproduction in the single-celled photosynthetic alga, Chlamydomonas reinhardtii . Specifically, we show that RLS1 mutants are unable to properly suppress their reproduction in phosphate-deprived conditions. Although these mutants have an immediate reproductive advantage relative to the parental strain, their long-term survival is negatively affected. Our data suggest that RLS1 is a bona fide life-history trade-off gene that suppresses immediate reproduction and ensures survival by downregulating photosynthesis in limiting environments, as part of the general acclimation response to nutrient deprivation in photosynthetic organisms.

Chlamydomonas reinhardtii undergoes conditional senescence when grown in batch culture due to nutrient limitation. Here, we explored plastid and photo-physiological adaptations in Chlamydomonas reinhardtii during a long-term ageing experiment by methodically sampling them over 22 weeks. Following exponential growth, Chlamydomonas entered an extended declining growth phase where cells continued to divide, although at a lower rate. Ultimately, this ongoing division was fueled by the recycling of macromolecules that was obvious in the rapidly declining protein and chlorophyll content in the cell during this phase. This process was sufficient to maintain a high level of cell viability as the culture entered stationary phase. Beyond that the cell viability starts to plummet. During the turnover of macromolecules after exponential growth that saw RuBisCO levels drop, the LHCII antenna was relatively stable. This, along with the upregulation of the light stress-related proteins (LHCSR), contributes to an efficient energy dissipation mechanism to protect the ageing cells from photooxidative stress during the senescence process. Ultimately, viability dropped to about 7% at 22 weeks in a batch culture. We anticipate that this research will help further understand the various acclimation strategies carried out by Chlamydomonas to maximize survival under conditional senescence., (© 2021. Crown.)

Microalgae can respond to long-term increases in light intensity by altering the concentration of photosynthetic complexes. Under active growth, the ability of Chlamydomonas reinhardtii to acclimate to excess light is dependent on cell division to reduce the concentration of photosynthetic complexes. But, in batch culture, cells eventually reach stationary phase where their ability to divide is limited; this should impact their capacity to undergo photoacclimation. Our goal is to dissect excess-light responses as cells approach stationary phase and to determine how the strategies of photoacclimation differ compared to cells in the exponential-growth phase. In this study, cultures exited exponential growth and transitioned into a declining growth phase (DGP), where cells continued a slow rate of growth for the next seven days in both low (LL) and high-light (HL). During this period, both cultures experience a conditional senescence-related decline in chlorophyll levels. Under HL, however, the senescing cultures have a rapid decline in PSII reaction centres, maintain a stable concentration of LHCII antenna, rapidly increase LHCSR levels, and have a sustained increase in Fo/Fm. Collectively this implies that the remaining antenna act as pH-dependent, quenching centres, presumably to protect the senescing chloroplast against HL. We discovered that acclimating to HL post-exponential phase involves active degradation that is intertwined with the normal senescence process that allowed for a limited rate of cell division., (Crown Copyright © 2020. Published by Elsevier Masson SAS. All rights reserved.)

Bigelowiella natans is a marine chlorarachniophyte whose plastid was acquired secondarily via endosymbiosis with a green alga. During plastid evolution, the photosynthetic endosymbiont would have integrated with the host metabolic pathways. This would require the evolution and coordination of strategies to cope with changes in light intensity that includes changes in the expression of both endosymbiont and host-derived genes. To investigate the transcriptional response to light intensity in chlorarachniophytes, we conducted an RNA-seq experiment to identify differentially expressed genes following a 4-h shift to high or very-low light. A shift to high light altered the expression of over 2,000 genes, many involved with photosynthesis, PSII assembly, primary metabolism, and reactive-oxygen scavenging. These changes are an attempt to optimize photosynthesis and increase energy sinks for excess reductant, while minimizing photooxidative stress. A transfer to very-low light resulted in a lower photosynthetic performance and metabolic alteration, reflecting an energy-limited state. Genes located on the nucleomorph, the vestigial nucleus in the plastid, had few changes in expression in either light treatment, indicating this organelle has relinquished most transcriptional control to the nucleus. Overall, during plastid origin, both host and transferred endosymbiont genes evolved a harmonized transcriptional network to respond to a classic photosynthetic stress., (© 2018 International Society of Protistologists.)

Bigelowiella natans is a mixotrophic flagellate and member of the chlorarachniophytes (Rhizaria), whose plastid is derived from a green algal endosymbiont. With the completion of the B. natans nuclear genome we are able to begin the analysis of the structure, function and evolution of the photosynthetic apparatus. B. natans has undergone substantial changes in photosystem structure during the evolution of the plastid from a green alga. While Photosystem II (PSII) composition is well conserved, Photosystem I (PSI) composition has undergone a dramatic reduction in accessory protein subunits. Coinciding with these changes, there was a loss of green algal LHCI orthologs while the PSII-like antenna system has the expected green algal-like proteins (encoded by genes Lhcbm1-8, Lhcb4). There are also a collection of LHCX-like proteins, which are commonly associated with stramenopiles and other eukaryotes with red-algal derived plastids, along with two other unique classes of LHCs- LHCY and LHCZ- whose function remains cryptic. To understand the regulation of the LHC gene family as an initial probe of function, we conducted an RNA-seq experiment under a short-term, high-light (HL) and low-light stress. The most abundant LHCII transcript (Lhcbm6) plus two other LHCBM types (Lhcbm1, 2) were down regulated under HL and up-regulated following a shift to very-low light (VL), as is common in antenna specializing in light harvesting. Many of the other LHCII and LHCY genes had a small, but significant increase in HL and most were only moderately affected under VL. The LHCX and LHCZ genes, however, had a strong up-regulation under HL-stress and most declined under VL, suggesting that they primarily have a role in photoprotection. This contrasts to the LHCY family that is only moderately responsive to light and a much higher basal level of expression, despite being within the LHCSR/LHCX clade. The expression of LHCX/Z proteins under HL-stress may be related to the induction of long-term, non-photochemical quenching mechanisms., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

The lateral transfer of photosynthesis between kingdoms through endosymbiosis is among the most spectacular examples of evolutionary innovation. Euglena, which acquired a chloroplast indirectly through an endosymbiosis with a green alga, represents such an example. As with other endosymbiont-derived plastids from eukaryotes, there are additional membranes that surround the organelle, of which Euglena has three. Thus, photosynthetic genes that were transferred from the endosymbiont to the host nucleus and whose proteins are required in the new plastid, are now faced with targeting and plastid import challenges. Early immunoelectron microscopy data suggested that the light-harvesting complexes, photosynthetic proteins in the thylakoid membrane, are post-translationally targeted to the plastid via the Golgi apparatus, an unexpected discovery at the time. Proteins targeted to the Euglena plastid have complex, bipartite presequences that direct them into the endomembrane system, through the Golgi apparatus and ultimately on to the plastid, presumably via transport vesicles. From transcriptome sequencing, dozens of plastid-targeted proteins were identified, leading to the identification of two different presequence structures. Both have an amino terminal signal peptide followed by a transit peptide for plastid import, but only one of the two classes of presequences has a third domain-the stop transfer sequence. This discovery implied two different transport mechanisms; one where the protein was fully inserted into the lumen of the ER and another where the protein remains attached to, but effectively outside, the endomembrane system. In this review, we will discuss the biochemical and bioinformatic evidence for plastid targeting, discuss the evolution of the targeting system, and ultimately provide a working model for the targeting and import of proteins into the plastid of Euglena.

There are several well-described acclimation responses to excess light in green algae but the effect on metabolism has not been thoroughly investigated. This study examines the metabolic changes during photoacclimation to high-light (HL) stress in Chlamydomonas reinhardtii using nuclear magnetic resonance and mass spectrometry. Using principal component analysis, a clear metabolic response to HL intensity was observed on global metabolite pools, with major changes in the levels of amino acids and related nitrogen metabolites. Amino acid pools increased during short-term photoacclimation, but were especially prominent in HL-acclimated cultures. Unexpectedly, we observed an increase in mitochondrial metabolism through downstream photorespiratory pathways. The expression of two genes encoding key enzymes in the photorespiratory pathway, glycolate dehydrogenase and malate synthase, were highly responsive to the HL stress. We propose that this pathway contributes to metabolite pools involved in nitrogen assimilation and may play a direct role in photoacclimation. Our results suggest that primary and secondary metabolism is highly pliable and plays a critical role in coping with the energetic imbalance during HL exposure and a necessary adjustment to support an increased growth rate that is an effective energy sink for the excess reducing power generated during HL stress., (© 2013 John Wiley & Sons Ltd.)

The mechanisms of microalgal senescence may play an important role in nutrient recycling and enhanced survival. However, the aging physiology of microalgae is an understudied phenomenon. To investigate the patterns of conditional senescence in Chlamydomonas reinhardtii P. A. Dangeard, we used a cell wall-less strain, transformed with a reporter gene to infer changes in photosynthetic gene expression. We examined plastid ultrastructure, photosynthetic function, and photoprotective mechanisms during aging in batch cultures. LHCII transcription levels decreased before the population entered stationary phase, and the characteristic transcriptional light-shift response was lost. A decline in photosynthetic proteins with a concomitant increase in the photoprotective protein, LHCSR, was observed over time. However, nonphotochemical quenching remained stable during growth and stationary phase, and then declined as alternative quenching mechanisms were up-regulated. Photosynthetic efficiency declined, while Fv/Fm remained stable until the death phases. As the culture progressed through stationary phase, disorganization of the chloroplast was observed along with an increase in cytoplasmic oil bodies. We also observed a partial recovery of function and proteins during the final death phase, and attribute this to the release of nutrients into the medium from cell lysis and/or active secretion while cells were senescing. Allowing open gas exchange resulted in high levels of sustained starch production and maintained maximum cell density, prolonging the stationary phase., (© 2013 Phycological Society of America.)

Cryptophyte and chlorarachniophyte algae are transitional forms in the widespread secondary endosymbiotic acquisition of photosynthesis by engulfment of eukaryotic algae. Unlike most secondary plastid-bearing algae, miniaturized versions of the endosymbiont nuclei (nucleomorphs) persist in cryptophytes and chlorarachniophytes. To determine why, and to address other fundamental questions about eukaryote-eukaryote endosymbiosis, we sequenced the nuclear genomes of the cryptophyte Guillardia theta and the chlorarachniophyte Bigelowiella natans. Both genomes have >21,000 protein genes and are intron rich, and B. natans exhibits unprecedented alternative splicing for a single-celled organism. Phylogenomic analyses and subcellular targeting predictions reveal extensive genetic and biochemical mosaicism, with both host- and endosymbiont-derived genes servicing the mitochondrion, the host cell cytosol, the plastid and the remnant endosymbiont cytosol of both algae. Mitochondrion-to-nucleus gene transfer still occurs in both organisms but plastid-to-nucleus and nucleomorph-to-nucleus transfers do not, which explains why a small residue of essential genes remains locked in each nucleomorph.

The primary endosymbiotic origin of the plastid in eukaryotes more than 1 billion years ago led to the evolution of algae and plants. We analyzed draft genome and transcriptome data from the basally diverging alga Cyanophora paradoxa and provide evidence for a single origin of the primary plastid in the eukaryote supergroup Plantae. C. paradoxa retains ancestral features of starch biosynthesis, fermentation, and plastid protein translocation common to plants and algae but lacks typical eukaryotic light-harvesting complex proteins. Traces of an ancient link to parasites such as Chlamydiae were found in the genomes of C. paradoxa and other Plantae. Apparently, Chlamydia-like bacteria donated genes that allow export of photosynthate from the plastid and its polymerization into storage polysaccharide in the cytosol.

Chlorarachniophytes are unicellular marine algae with plastids (chloroplasts) of secondary endosymbiotic origin. Chlorarachniophyte cells retain the remnant nucleus (nucleomorph) and cytoplasm (periplastidial compartment, PPC) of the green algal endosymbiont from which their plastid was derived. To characterize the diversity of nucleus-encoded proteins targeted to the chlorarachniophyte plastid, nucleomorph, and PPC, we isolated plastid-nucleomorph complexes from the model chlorarachniophyte Bigelowiella natans and subjected them to high-pressure liquid chromatography-tandem mass spectrometry. Our proteomic analysis, the first of its kind for a nucleomorph-bearing alga, resulted in the identification of 324 proteins with 95% confidence. Approximately 50% of these proteins have predicted bipartite leader sequences at their amino termini. Nucleus-encoded proteins make up >90% of the proteins identified. With respect to biological function, plastid-localized light-harvesting proteins were well represented, as were proteins involved in chlorophyll biosynthesis. Phylogenetic analyses revealed that many, but by no means all, of the proteins identified in our proteomic screen are of apparent green algal ancestry, consistent with the inferred evolutionary origin of the plastid and nucleomorph in chlorarachniophytes.

Eukaryotes acquired photosynthetic metabolism over a billion years ago, and during that time the light-harvesting antennae have undergone significant structural and functional divergence. The antenna systems are generally used to harvest and transfer excitation energy into the reaction centers to drive photosynthesis, but also have the dual role of energy dissipation. Phycobilisomes formed the first antenna system in oxygenic photoautotrophs, and this soluble protein complex continues to be the dominant antenna in extant cyanobacteria, glaucophytes, and red algae. However, phycobilisomes were lost multiple times during eukaryotic evolution in favor of a thylakoid membrane-integral light-harvesting complex (LHC) antenna system found in the majority of eukaryotic taxa. While photosynthesis spread across different eukaryotic kingdoms via endosymbiosis, the antenna systems underwent extensive modification as photosynthetic groups optimized their light-harvesting capacity and ability to acclimate to changing environmental conditions. This review discusses the different classes of LHCs within photosynthetic eukaryotes and examines LHC diversification in different groups in a structural and functional context.

Light-harvesting-like (LIL) proteins are low-molecular-mass membrane proteins related to the light-harvesting complexes, which form the dominant antenna system in most photosynthetic eukaryotes. To analyze the LIL protein family, we mined a number of publicly available databases to identify members of this family in a broad range of organisms. LIL proteins are diverse, having one to three predicted transmembrane helices. One- and two-helix LIL proteins were found in all the major photosynthetic eukaryote lineages (glaucophytes, red algae, and green algae) and are particularly well conserved in the green algae and land plants. In most cases, however, these proteins are not conserved between major lineages, and in some cases appear to have evolved independently. Three-helix LIL proteins are well conserved within the gymnosperms and angiosperms, but are much more divergent, and have been duplicated multiple times, in the green algae and bryophytes. We also identified a novel LIL protein in two Micromonas strains that contains a fourth hydrophobic region. This analysis identifies conserved members of the LIL protein family, signifying their importance to photosynthetic eukaryotes. It also indicates that classification of these proteins based on structural characteristics alone inadequately reflects the evolutionary history observed in this complex protein family.

Chlamydomonas reinhardtii, a unicellular green alga, has been used in bioassay detection of a variety of toxic compounds such as pesticides and toxic metals, but mainly using liquid culture systems. In this study, an algal lawn-agar system for semi-quantitative bioassay of herbicidal activities has been developed. Sixteen different herbicides belonging to 11 different categories were applied to paper disks and placed on green alga lawns in Petri dishes. Presence of herbicide activities was indicated by clearing zones around the paper disks on the lawn 2-3 days after application. The different groups of herbicides induced clearing zones of variable size that depended on the amount, mode of action, and chemical properties of the herbicides applied to the paper disks. This simple, paper-disk-algal system may be used to detect the presence of herbicides in water samples and act as a quick and inexpensive semi-quantitative screening for assessing herbicide contamination.

Background: The sulfate assimilation pathway is present in photosynthetic organisms, fungi, and many bacteria, providing reduced sulfur for the synthesis of cysteine and methionine and a range of other metabolites. In photosynthetic eukaryotes sulfate is reduced in the plastids whereas in aplastidic eukaryotes the pathway is cytosolic. The only known exception is Euglena gracilis, where the pathway is localized in mitochondria. To obtain an insight into the evolution of the sulfate assimilation pathway in eukaryotes and relationships of the differently compartmentalized isoforms we determined the locations of the pathway in lineages for which this was unknown and performed detailed phylogenetic analyses of three enzymes involved in sulfate reduction: ATP sulfurylase (ATPS), adenosine 5'-phosphosulfate reductase (APR) and sulfite reductase (SiR)., Results: The inheritance of ATPS, APR and the related 3'-phosphoadenosine 5'-phosphosulfate reductase (PAPR) are remarkable, with multiple origins in the lineages that comprise the opisthokonts, different isoforms in chlorophytes and streptophytes, gene fusions with other enzymes of the pathway, evidence a eukaryote to prokaryote lateral gene transfer, changes in substrate specificity and two reversals of cellular location of host- and endosymbiont-originating enzymes. We also found that the ATPS and APR active in the mitochondria of Euglena were inherited from its secondary, green algal plastid., Conclusion: Our results reveal a complex history for the enzymes of the sulfate assimilation pathway. Whilst they shed light on the origin of some characterised novelties, such as a recently described novel isoform of APR from Bryophytes and the origin of the pathway active in the mitochondria of Euglenids, the many distinct and novel isoforms identified here represent an excellent resource for detailed biochemical studies of the enzyme structure/function relationships.

Light-harvesting complexes (LHCs) are a superfamily of chlorophyll- and carotenoid-binding proteins that are responsible for the capture of light energy and its transfer to the photosynthetic reaction centers. Unlike those of most eukaryotes, the LHCs of Euglena gracilis are translated from large mRNAs, producing polyprotein precursors consisting of multiple concatenated LHC subunits that are separated by conserved decapeptide linkers. These precursors are posttranslationally targeted to the chloroplast and cleaved into individual proteins. We analyzed expressed sequence tags from Euglena to further characterize the structural features of the LHC polyprotein-coding genes and to examine the evolution of this multigene family. Of the 19 different LHC transcriptional units we detected, 17 encoded polyproteins composed of both tandem and nontandem repeats of LHC subunits; organizations that likely occurred through unequal crossing-over. Of the 2 nonpolyprotein-encoding LHC transcripts detected, 1 evolved from the truncation of a polyprotein-coding gene. Duplication of LHC polyprotein-coding genes was particularly important in the LHCI gene family where multiple paralogous sequences were detected. Intriguingly, several of the individual LHC-coding subunits both within and between transcriptional units appeared to be evolving in concert, suggesting that gene conversion has been a significant mechanism for LHC evolution in Euglena.

Background: Lateral gene transfer is increasingly invoked to explain phylogenetic results that conflict with our understanding of organismal relationships. In eukaryotes, the most common observation interpreted in this way is the appearance of a bacterial gene (one that is not clearly derived from the mitochondrion or plastid) in a eukaryotic nuclear genome. Ideally such an observation would involve a single eukaryote or a small group of related eukaryotes encoding a gene from a specific bacterial lineage., Results: Here we show that several apparently simple cases of lateral transfer are actually more complex than they originally appeared: in these instances we find that two or more distantly related eukaryotic groups share the same bacterial gene, resulting in a punctate distribution. Specifically, we describe phylogenies of three core carbon metabolic enzymes: transketolase, glyceraldehyde-3-phosphate dehydrogenase and ribulose-5-phosphate-3-epimerase. Phylogenetic trees of each of these enzymes includes a strongly-supported clade consisting of several eukaryotes that are distantly related at the organismal level, but whose enzymes are apparently all derived from the same lateral transfer. With less sampling any one of these examples would appear to be a simple case of bacterium-to-eukaryote lateral transfer; taken together, their evolutionary histories cannot be so simple. The distributions of these genes may represent ancient paralogy events or genes that have been transferred from bacteria to an ancient ancestor of the eukaryotes that retain them. They may alternatively have been transferred laterally from a bacterium to a single eukaryotic lineage and subsequently transferred between distantly related eukaryotes., Conclusion: Determining how complex the distribution of a transferred gene is depends on the sampling available. These results show that seemingly simple cases may be revealed to be more complex with greater sampling, suggesting many bacterial genes found in eukaryotic genomes may have a punctate distribution.

The light-harvesting complexes (LHCs) of land plants and green algae have essential roles in light capture and photoprotection. Though the functional diversity of the individual LHC proteins are well described in many land plants, the extent of this family in the majority of green algal groups is unknown. To examine the evolution of the chlorophyll a/b antennae system and to infer its ancestral state, we initiated several expressed sequence tag projects from a taxonomically broad range of chlorophyll a/b-containing protists. This included representatives from the Ulvophyceae (Acetabularia acetabulum), the Mesostigmatophyceae (Mesostigma viride), and the Prasinophyceae (Micromonas sp.), as well as one representative from each of the Euglenozoa (Euglena gracilis) and Chlorarachniophyta (Bigelowiella natans), whose plastids evolved secondarily from a green alga. It is clear that the core antenna system was well developed prior to green algal diversification and likely consisted of the CP29 (Lhcb4) and CP26 (Lhcb5) proteins associated with photosystem II plus a photosystem I antenna composed of proteins encoded by at least Lhca3 and two green algal-specific proteins encoded by the Lhca2 and 9 genes. In organisms containing secondary plastids, we found no evidence for orthologs to the plant/algal antennae with the exception of CP29. We also identified PsbS homologs in the Ulvophyceae and the Prasinophyceae, indicating that this distinctive protein appeared prior to green algal diversification. This analysis provides a snapshot of the antenna systems in diverse green algae, and allows us to infer the changing complexity of the antenna system during green algal evolution.

The plastid of Euglena gracilis was acquired secondarily through an endosymbiotic event with a eukaryotic green alga, and as a result, it is surrounded by a third membrane. This membrane complexity raises the question of how the plastid proteins are targeted to and imported into the organelle. To further explore plastid protein targeting in Euglena, we screened a total of 9,461 expressed sequence tag (EST) clusters (derived from 19,013 individual ESTs) for full-length proteins that are plastid localized to characterize their targeting sequences and to infer potential modes of translocation. Of the 117 proteins identified as being potentially plastid localized whose N-terminal targeting sequences could be inferred, 83 were unique and could be classified into two major groups. Class I proteins have tripartite targeting sequences, comprising (in order) an N-terminal signal sequence, a plastid transit peptide domain, and a predicted stop-transfer sequence. Within this class of proteins are the lumen-targeted proteins (class IB), which have an additional hydrophobic domain similar to a signal sequence and required for further targeting across the thylakoid membrane. Class II proteins lack the putative stop-transfer sequence and possess only a signal sequence at the N terminus, followed by what, in amino acid composition, resembles a plastid transit peptide. Unexpectedly, a few unrelated plastid-targeted proteins exhibit highly similar transit sequences, implying either a recent swapping of these domains or a conserved function. This work represents the most comprehensive description to date of transit peptides in Euglena and hints at the complex routes of plastid targeting that must exist in this organism.

When challenged with excess light, the green alga Chlamydomonas reinhardtii responds, in part, by down-regulating light harvesting capacity at Photosystem II while concomitantly reorganising cellular metabolism to increase sink capacity. We examined the role of translational control during different stages of photoacclimation by analysing polysome profiles of two different light-harvesting complex (LHC) genes encoding a major LHCII component (Lhcbm) and CP29 (Lhcb4) plus iron superoxide dismutase (FeSOD), and through measurement of protein synthesis by in vivo labelling. Within two hours following transfer of low-light (LL) acclimated cultures into high-light (HL), Lhcbm transcripts are off-loaded from polysomes indicating a decline in translational initiation. Lhcbm translational repression at two hours was specific since FeSOD mRNA remained associated with polysomes and in vivo labelling showed an increase in overall protein synthesis. After 4 hours HL exposure, however, a global disassembly of polysomes was detected suggesting a general decline in translational initiation. Interestingly, the decrease in polysomes coincides with maximal FeSOD transcript levels emphasizing that transcript profiles do not always accurately reflect gene expression. Within 8 hours after LL to HL shift, polysomes reform and all transcripts examined are loaded back onto polysomes. Disassembly of polysomes was mimicked by hydrogen peroxide application to non-shifted cultures suggesting that production of hydrogen peroxide due to HL-stress may effect the general decline in polysomes observed during HL acclimation.

Recent advances in resolving the tree of eukaryotes are converging on a model composed of a few large hypothetical 'supergroups', each comprising a diversity of primarily microbial eukaryotes (protists, or protozoa and algae). The process of resolving the tree involves the synthesis of many kinds of data, including single-gene trees, multigene analyses, and other kinds of molecular and structural characters. Here, we review the recent progress in assembling the tree of eukaryotes, describing the major evidence for each supergroup, and where gaps in our knowledge remain. We also consider other factors emerging from phylogenetic analyses and comparative genomics, in particular lateral gene transfer, and whether such factors confound our understanding of the eukaryotic tree.

Novel eukaryotic chlorophyll-carotenoid proteins have evolved at least twice following the origin of the plastid and include the widely distributed integral membrane light-harvesting complexes (LHCs) and the dinoflagellate-specific soluble peridinin-chlorophyll proteins. In the glaucophytes, homologs of these proteins are reportedly absent. We have identified a novel carotenoid-rich protein (CRP) in the glaucophyte Cyanophora paradoxa that is 28 kDa and immunologically related to the family of LHCs. CRP is associated with the thylakoid membrane, though it can be removed by stringent washes, suggesting that there are probably significant structural differences between CRP and the LHCs. CRP co-localizes with a zeaxanthin-rich thylakoid membrane fraction that also contains beta-carotene, chlorophyll and an unidentified carotenoid. Despite this, we found no evidence for carotenoid-chlorophyll energy transfer in the isolated complex, suggesting that light harvesting may not be a primary function. The presence of CRP in C. paradoxa is evidence for the evolution of a novel pigment-binding protein in the glaucophytes.

We identify four novel DNA-binding complexes in the nuclear-encoded Lhcb1 promoter of the chlorophyte alga Dunaliella tertiolecta that are regulated by photosynthetic pathways in the plastid. The binding activities of three of the complexes were positively correlated with time-dependent changes in Lhcb1 transcript abundance, implicating their roles as transcriptional enhancers in a retrograde signal transduction pathway. Using a combination of inhibitors, uncouplers, and antimycin A, and by following the kinetic pattern of gene regulation, we infer two different sensors in the signal transduction pathway. On short time scales of 0.5 to about 4 h, the transthylakoid membrane potential appears to be a critical determinant of gene expression, whereas on time scales of 8 h or longer, the redox state of the plastoquinone pool becomes increasingly more important. The differentiation of these two types of signals was observed in parallel effects on gene transcription and on the patterns of DNA-binding activities in the Lhcb1 promoter. These signals appear to be transduced at the nuclear level via a coordinated ensemble of DNA-binding complexes located between -367 and -188 bp from the start codon of the gene. The regulation of these elements allows the cell to up- or down-regulate the expression on Lhcb1 in response to changes in irradiance.

Cryptomonads and chlorarachniophytes acquired photosynthesis independently by engulfing and retaining eukaryotic algal cells. The nucleus of the engulfed cells (known as a nucleomorph) is much reduced and encodes only a handful of the numerous essential plastid proteins normally encoded by the nucleus of chloroplast-containing organisms. In cryptomonads and chlorarachniophytes these proteins are thought to be encoded by genes in the secondary host nucleus. Genes for these proteins were potentially transferred from the nucleomorph (symbiont nucleus) to the secondary host nucleus; nucleus to nucleus intracellular gene transfers. We isolated complementary DNA clones (cDNAs) for chlorophyll-binding proteins from a cryptomonad and a chlorarachniophyte. In each organism these genes reside in the secondary host nuclei, but phylogenetic evidence, and analysis of the targeting mechanisms, suggest the genes were initially in the respective nucleomorphs (symbiont nuclei). Implications for origins of secondary endosymbiotic algae are discussed.

The light-harvesting complexes (LHCs) are a superfamily of chlorophyll-binding proteins present in all photosynthetic eukaryotes. The Lhc genes are nuclear-encoded, yet the pigment-protein complexes are localized to the thylakoid membrane and provide a marker to follow the evolutionary paths of plastids with different pigmentation. The LHCs are divided into the chlorophyll a/b-binding proteins of the green algae, euglenoids, and higher plants and the chlorophyll a/c-binding proteins of various algal taxa. This work examines the phylogenetic position of the LHCs from three additional taxa: the rhodophytes, the cryptophytes, and the chlorarachniophytes. Phylogenetic analysis of the LHC sequences provides strong statistical support for the clustering of the rhodophyte and cryptomonad LHC sequences within the chlorophyll a/c-binding protein lineage, which includes the fucoxanthin-chlorophyll proteins (FCP) of the heterokonts and the intrinsic peridinin-chlorophyll proteins (iPCP) of the dinoflagellates. These associations suggest that plastids from the heterokonts, haptophytes, cryptomonads, and the dinoflagellate, Amphidinium, evolved from a red algal-like ancestor. The Chlorarachnion LHC is part of the chlorophyll a/b-binding protein assemblage, consistent with pigmentation, providing further evidence that its plastid evolved from a green algal secondary endosymbiosis. The Chlorarachnion LHC sequences cluster with the green algal LHCs that are predominantly associated with photosystem II (LHCII). This suggests that the green algal endosymbiont that evolved into the Chlorarachnion plastid was acquired following the emergence of distinct LHCI and LHCII complexes.

A fucoxanthin-chlorophyll protein (FCP) cDNA from the raphidophyte Heterosigma carterae encodes a 210-amino acid polypeptide that has similarity to other FCPs and to the chlorophyll a/b-binding proteins (CABs) of terrestrial plants and green algae. The putative transit sequence has characteristics that resemble a signal sequence. The Heterosigma fcp genes are part of a large multigene family which includes members encoding at least two significantly different polypeptides (Fcp1, Fcp2). Comparison of the FCP sequences to the recently determined three-dimensional structure of the pea LHC II complex indicates that many of the key amino acids thought to participate in the binding of chlorophyll and the formation of complex-stabilizing ionic interactions are well conserved. Phylogenetic analyses of sequences of light-harvesting proteins shows that the FCPs of several chromophyte phyla form a natural group separate from the intrinisic peridinin-chlorophyll proteins (iPCPs) of the dinoflagellates: Although the FCP and CAB genes shared a common ancestor, these lineages diverged from each other prior to the separation of the CAB LHC I and LHC II sequences in the green algae and terrestrial plants.

The chlorophyll-carotenoid binding proteins responsible for absorption and conversion of light energy in oxygen-evolving photosynthetic organisms belong to two extended families: the Chl a binding core complexes common to cyanobacteria and all chloroplasts, and the nuclear-encoded light-harvesting antenna complexes of eukaryotic photosynthesizers (Chl a/b, Chl a/c, and Chl a proteins). There is a general consensus on polypeptide and pigment composition for higher plant pigment proteins. These are reviewed and compared with pigment proteins of chlorophyte, rhodophyte, and chromophyte algae. Major advances have been the determination of the structures of LHCII (major Chl a/b complex of higher plants), cyanobacterial Photosystem I, and the peridinen-Chl a protein of dinoflagellates to atomic resolution. Better isolation methods, improved transformation procedures, and the availability of molecular structure models are starting to provide insights into the pathways of energy transfer and the macromolecular organization of thylakoid membranes.

A 0.8 kb AvaI/SmaI fragment of the plastid genome of the chlorophyll c-containing alga Cryptomonas phi encompassing the rRNA spacer region and flanking genes has been cloned and sequenced. The spacer region between the 16S and 23S rRNA genes is 275 base pairs long, one of the shortest yet reported, and it contains uninterrupted genes for tRNA(Ile) and tRNA(Ala) separated by only two base pairs. The coding regions for tRNAs and rRNAs have been compared with those from cyanobacteria, land plants and other algae and the possible evolutionary relationships discussed.

The gene for the small subunit of ribulose-1,5-bisphosphate carboxylase (Rubisco) is located in the large single-copy region of the plastid genome of the chlorophyll c-containing alga Cryptomonas phi. The coding sequence is 417 base pairs long, encoding a protein of 139 amino acids, considerably longer than most other small subunit proteins. It is found 83 base pairs downstream from the gene for the large subunit and is cotranscribed with it. An 18 base pair perfect inverted repeat is located 8 base pairs beyond the termination codon. Sequence analysis shows the gene to be more closely related to cyanobacterial and cyanelle small-subunit genes than to those of green algae or land plants. This is the first reported sequence of a Rubisco small-subunit gene which is plastid-encoded and it exhibits a number of unique features. The derived amino acid sequence shows extensive similarity to a partial amino acid sequence from a brown alga, indicating that this gene will be of major interest as a probe for the small subunit genes in other algae and for determining possible evolutionary ancestors of algal plastids.