RESUMO
Five versions of the Chlamydomonas reinhardtii reference genome have been produced over the last two decades. Here we present version 6, bringing significant advances in assembly quality and structural annotations. PacBio-based chromosome-level assemblies for two laboratory strains, CC-503 and CC-4532, provide resources for the plus and minus mating-type alleles. We corrected major misassemblies in previous versions and validated our assemblies via linkage analyses. Contiguity increased over ten-fold and >80% of filled gaps are within genes. We used Iso-Seq and deep RNA-seq datasets to improve structural annotations, and updated gene symbols and textual annotation of functionally characterized genes via extensive manual curation. We discovered that the cell wall-less classical reference strain CC-503 exhibits genomic instability potentially caused by deletion of the helicase RECQ3, with major structural mutations identified that affect >100 genes. We therefore present the CC-4532 assembly as the primary reference, although this strain also carries unique structural mutations and is experiencing rapid proliferation of a Gypsy retrotransposon. We expect all laboratory strains to harbor gene-disrupting mutations, which should be considered when interpreting and comparing experimental results. Collectively, the resources presented here herald a new era of Chlamydomonas genomics and will provide the foundation for continued research in this important reference organism.
Assuntos
Chlamydomonas reinhardtii , Chlamydomonas , Chlamydomonas/genética , Genômica/métodos , Mutação/genética , Reprodução , Chlamydomonas reinhardtii/genéticaRESUMO
Marine algae are responsible for half of the world's primary productivity, but this critical carbon sink is often constrained by insufficient iron. One species of marine algae, Dunaliella tertiolecta, is remarkable for its ability to maintain photosynthesis and thrive in low-iron environments. A related species, Dunaliella salina Bardawil, shares this attribute but is an extremophile found in hypersaline environments. To elucidate how algae manage their iron requirements, we produced high-quality genome assemblies and transcriptomes for both species to serve as a foundation for a comparative multiomics analysis. We identified a host of iron-uptake proteins in both species, including a massive expansion of transferrins and a unique family of siderophore-iron-uptake proteins. Complementing these multiple iron-uptake routes, ferredoxin functions as a large iron reservoir that can be released by induction of flavodoxin. Proteomic analysis revealed reduced investment in the photosynthetic apparatus coupled with remodeling of antenna proteins by dramatic iron-deficiency induction of TIDI1, which is closely related but identifiably distinct from the chlorophyll binding protein, LHCA3. These combinatorial iron scavenging and sparing strategies make Dunaliella unique among photosynthetic organisms.
Assuntos
Clorofíceas , Extremófilos , Ferro/metabolismo , Multiômica , Proteômica , Fotossíntese , Proteínas/metabolismoRESUMO
Lysosome-related organelles (LROs) are a class of heterogeneous organelles conserved in eukaryotes that primarily play a role in storage and secretion. An important function of LROs is to mediate metal homeostasis. Chlamydomonas reinhardtii is a model organism for studying metal ion metabolism; however, structural and functional analyses of LROs in C. reinhardtii are insufficient. Here, we optimized a method for purifying these organelles from 2 populations of cells: stationary phase or overloaded with iron. The morphology, elemental content, and lysosomal activities differed between the 2 preparations, even though both have phosphorus and metal ion storage functions. LROs in stationary phase cells had multiple non-membrane-bound polyphosphate granules to store phosphorus. Those in iron-overloaded cells were similar to acidocalcisomes (ACs), which have a boundary membrane and contain 1 or 2 large polyphosphate granules to store more phosphorus. We established a method for quantifying the capacity of LROs to sequester individual trace metals. Based on a comparative proteomic analysis of these 2 types of LROs, we present a comprehensive AC proteome and identified 113 putative AC proteins. The methods and protein inventories provide a framework for studying the biogenesis and modification of LROs and the mechanisms by which they participate in regulating metal ion metabolism.
Assuntos
Chlamydomonas , Chlamydomonas/metabolismo , Proteômica , Organelas/metabolismo , Lisossomos/metabolismo , Polifosfatos/metabolismo , Fósforo/metabolismoRESUMO
Low iron (Fe) bioavailability can limit the biosynthesis of Fe-containing proteins, which are especially abundant in photosynthetic organisms, thus negatively affecting global primary productivity. Understanding cellular coping mechanisms under Fe limitation is therefore of great interest. We surveyed the temporal responses of Chlamydomonas (Chlamydomonas reinhardtii) cells transitioning from an Fe-rich to an Fe-free medium to document their short and long-term adjustments. While slower growth, chlorosis and lower photosynthetic parameters are evident only after one or more days in Fe-free medium, the abundance of some transcripts, such as those for genes encoding transporters and enzymes involved in Fe assimilation, change within minutes, before changes in intracellular Fe content are noticeable, suggestive of a sensitive mechanism for sensing Fe. Promoter reporter constructs indicate a transcriptional component to this immediate primary response. With acetate provided as a source of reduced carbon, transcripts encoding respiratory components are maintained relative to transcripts encoding components of photosynthesis and tetrapyrrole biosynthesis, indicating metabolic prioritization of respiration over photosynthesis. In contrast to the loss of chlorophyll, carotenoid content is maintained under Fe limitation despite a decrease in the transcripts for carotenoid biosynthesis genes, indicating carotenoid stability. These changes occur more slowly, only after the intracellular Fe quota responds, indicating a phased response in Chlamydomonas, involving both primary and secondary responses during acclimation to poor Fe nutrition.
Assuntos
Chlamydomonas reinhardtii , Ferro , Fotossíntese , Ferro/metabolismo , Chlamydomonas reinhardtii/metabolismo , Chlamydomonas reinhardtii/fisiologia , Chlamydomonas reinhardtii/genética , Carotenoides/metabolismo , Clorofila/metabolismo , Chlamydomonas/metabolismo , Chlamydomonas/fisiologia , Regulação da Expressão Gênica de PlantasRESUMO
The unicellular green alga Chlamydomonas reinhardtii is a choice reference system for the study of photosynthesis and chloroplast metabolism, cilium assembly and function, lipid and starch metabolism, and metal homeostasis. Despite decades of research, the functions of thousands of genes remain largely unknown, and new approaches are needed to categorically assign genes to cellular pathways. Growing collections of transcriptome and proteome data now allow a systematic approach based on integrative co-expression analysis. We used a dataset comprising 518 deep transcriptome samples derived from 58 independent experiments to identify potential co-expression relationships between genes. We visualized co-expression potential with the R package corrplot, to easily assess co-expression and anti-correlation between genes. We extracted several hundred high-confidence genes at the intersection of multiple curated lists involved in cilia, cell division, and photosynthesis, illustrating the power of our method. Surprisingly, Chlamydomonas experiments retained a significant rhythmic component across the transcriptome, suggesting an underappreciated variable during sample collection, even in samples collected in constant light. Our results therefore document substantial residual synchronization in batch cultures, contrary to assumptions of asynchrony. We provide step-by-step protocols for the analysis of co-expression across transcriptome data sets from Chlamydomonas and other species to help foster gene function discovery.
Assuntos
Chlamydomonas reinhardtii/genética , Regulação da Expressão Gênica de Plantas , Redes Reguladoras de Genes , Arabidopsis/genética , Técnicas de Cultura Celular por Lotes , Núcleo Celular/genética , Núcleo Celular/metabolismo , Chlamydomonas reinhardtii/citologia , Cílios/genética , Ritmo Circadiano/genética , Genes de Plantas , Histonas/genética , Fotossíntese/genética , Proteínas Ribossômicas/genética , Volvox/genéticaRESUMO
The photosynthetic unicellular alga Chlamydomonas (Chlamydomonas reinhardtii) is a versatile reference for algal biology because of its ease of culture in the laboratory. Genomic and systems biology approaches have previously described transcriptome responses to environmental changes using bulk data, thus representing the average behavior from pools of cells. Here, we apply single-cell RNA sequencing (scRNA-seq) to probe the heterogeneity of Chlamydomonas cell populations under three environments and in two genotypes differing by the presence of a cell wall. First, we determined that RNA can be extracted from single algal cells with or without a cell wall, offering the possibility to sample natural algal communities. Second, scRNA-seq successfully separated single cells into nonoverlapping cell clusters according to their growth conditions. Cells exposed to iron or nitrogen deficiency were easily distinguished despite a shared tendency to arrest photosynthesis and cell division to economize resources. Notably, these groups of cells not only recapitulated known patterns observed with bulk RNA-seq but also revealed their inherent heterogeneity. A substantial source of variation between cells originated from their endogenous diurnal phase, although cultures were grown in constant light. We exploited this result to show that circadian iron responses may be conserved from algae to land plants. We document experimentally that bulk RNA-seq data represent an average of typically hidden heterogeneity in the population.
Assuntos
Chlamydomonas reinhardtii/citologia , Chlamydomonas reinhardtii/genética , Ritmo Circadiano/genética , Técnicas de Cultura Celular por Lotes , Parede Celular/genética , Chlamydomonas reinhardtii/fisiologia , Ferro/metabolismo , Nitrogênio/metabolismo , Proteínas de Plantas/genética , RNA de Plantas/isolamento & purificação , Análise de Sequência de RNA , Análise de Célula ÚnicaRESUMO
The acidocalcisome is an acidic organelle in the cytosol of eukaryotes, defined by its low pH and high calcium and polyphosphate content. It is visualized as an electron-dense object by transmission electron microscopy (TEM) or described with mass spectrometry (MS)-based imaging techniques or multimodal X-ray fluorescence microscopy (XFM) based on its unique elemental composition. Compared with MS-based imaging techniques, XFM offers the additional advantage of absolute quantification of trace metal content, since sectioning of the cell is not required and metabolic states can be preserved rapidly by either vitrification or chemical fixation. We employed XFM in Chlamydomonas reinhardtii to determine single-cell and organelle trace metal quotas within algal cells in situations of trace metal overaccumulation (Fe and Cu). We found up to 70% of the cellular Cu and 80% of Fe sequestered in acidocalcisomes in these conditions and identified two distinct populations of acidocalcisomes, defined by their unique trace elemental makeup. We utilized the vtc1 mutant, defective in polyphosphate synthesis and failing to accumulate Ca, to show that Fe sequestration is not dependent on either. Finally, quantitation of the Fe and Cu contents of individual cells and compartments via XFM, over a range of cellular metal quotas created by nutritional and genetic perturbations, indicated excellent correlation with bulk data from corresponding cell cultures, establishing a framework to distinguish the nutritional status of single cells.
Assuntos
Chlamydomonas reinhardtii/metabolismo , Processos Fototróficos/fisiologia , Oligoelementos/metabolismo , Chlamydomonas/metabolismo , Homeostase , Lisossomos/metabolismo , Microscopia Eletrônica de Transmissão/métodos , Organelas/metabolismo , Análise de Célula Única/métodos , Oligoelementos/análiseRESUMO
Polycistronic gene expression, common in prokaryotes, was thought to be extremely rare in eukaryotes. The development of long-read sequencing of full-length transcript isomers (Iso-Seq) has facilitated a reexamination of that dogma. Using Iso-Seq, we discovered hundreds of examples of polycistronic expression of nuclear genes in two divergent species of green algae: Chlamydomonas reinhardtii and Chromochloris zofingiensis Here, we employ a range of independent approaches to validate that multiple proteins are translated from a common transcript for hundreds of loci. A chromatin immunoprecipitation analysis using trimethylation of lysine 4 on histone H3 marks confirmed that transcription begins exclusively at the upstream gene. Quantification of polyadenylated [poly(A)] tails and poly(A) signal sequences confirmed that transcription ends exclusively after the downstream gene. Coexpression analysis found nearly perfect correlation for open reading frames (ORFs) within polycistronic loci, consistent with expression in a shared transcript. For many polycistronic loci, terminal peptides from both ORFs were identified from proteomics datasets, consistent with independent translation. Synthetic polycistronic gene pairs were transcribed and translated in vitro to recapitulate the production of two distinct proteins from a common transcript. The relative abundance of these two proteins can be modified by altering the Kozak-like sequence of the upstream gene. Replacement of the ORFs with selectable markers or reporters allows production of such heterologous proteins, speaking to utility in synthetic biology approaches. Conservation of a significant number of polycistronic gene pairs between C. reinhardtii, C. zofingiensis, and five other species suggests that this mechanism may be evolutionarily ancient and biologically important in the green algal lineage.
Assuntos
Clorófitas/genética , Regulação Bacteriana da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Fases de Leitura Aberta , Proteínas de Plantas/metabolismo , RNA Mensageiro/genética , Transcrição GênicaRESUMO
Chromatin modifications are epigenetic regulatory features with major roles in various cellular events, yet they remain understudied in algae. We interrogated the genome-wide distribution pattern of mono- and trimethylated histone H3 lysine 4 (H3K4) using chromatin-immunoprecipitation followed by deep-sequencing (ChIP-seq) during key phases of the Chlamydomonas cell cycle: early G1 phase, Zeitgeber Time 1 (ZT1), when cells initiate biomass accumulation, S/M phase (ZT13) when cells are replicating DNA and undergoing mitosis, and late G0 phase (ZT23) when they are quiescent. Tri-methylated H3K4 was predominantly enriched at transcription start sites of the majority of protein coding genes (85%). The likelihood of a gene being marked by H3K4me3 correlated with it being transcribed at some point during the life cycle but not necessarily by continuous active transcription, as exemplified by early zygotic genes, which may remain transcriptionally dormant for thousands of generations between sexual cycles. The exceptions to this rule were around 120 loci, some of which encode non-poly-adenylated transcripts, such as small nuclear RNAs and replication-dependent histones that had H3K4me3 peaks only when they were being transcribed. Mono-methylated H3K4 was the default state for the vast majority of histones that were bound outside of transcription start sites and terminator regions of genes. A small fraction of the genome that was depleted of any H3 lysine 4 methylation was enriched for DNA cytosine methylation and the genes within these DNA methylation islands were poorly expressed. Besides marking protein coding genes, H3K4me3 ChIP-seq data served also as a annotation tool for validation of hundreds of long non-coding RNA genes.
Assuntos
Chlamydomonas , RNA Longo não Codificante , Histonas/genética , Histonas/metabolismo , Lisina/metabolismo , Chlamydomonas/genética , Chlamydomonas/metabolismo , RNA Longo não Codificante/metabolismo , Metilação de DNA/genética , Cromatina/genética , CitosinaRESUMO
Even subtle modifications in growth conditions elicit acclimation responses affecting the molecular and elemental makeup of organisms, both in the laboratory and in natural habitats. We systematically explored the effect of temperature, pH, nutrient availability, culture density, and access to CO2 and O2 in laboratory-grown algal cultures on growth rate, the ionome, and the ability to accumulate Fe. We found algal cells accumulate Fe in alkaline conditions, even more so when excess Fe is present, coinciding with a reduced growth rate. Using a combination of Fe-specific dyes, X-ray fluorescence microscopy, and NanoSIMS, we show that the alkaline-accumulated Fe was intracellularly sequestered into acidocalcisomes, which are localized towards the periphery of the cells. At high photon flux densities, Zn and Ca specifically over-accumulate, while Zn alone accumulates at low temperatures. The impact of aeration was probed by reducing shaking speeds and changing vessel fill levels; the former increased the Cu quota of cultures, the latter resulted in a reduction in P, Ca, and Mn at low fill levels. Trace element quotas were also affected in the stationary phase, where specifically Fe, Cu, and Zn accumulate. Cu accumulation here depends inversely on the Fe concentration of the medium. Individual laboratory strains accumulate Ca, P, and Cu to different levels. All together, we identified a set of specific changes to growth rate, elemental composition, and the capacity to store Fe in response to subtle differences in culturing conditions of Chlamydomonas, affecting experimental reproducibility. Accordingly, we recommend that these variables be recorded and reported as associated metadata.
Assuntos
Chlamydomonas , Oligoelementos , Reprodutibilidade dos TestesRESUMO
The unicellular alga Chlamydomonas reinhardtii is a classical reference organism for studying photosynthesis, chloroplast biology, cell cycle control, and cilia structure and function. It is also an emerging model for studying sensory cilia, the production of high-value bioproducts, and in situ structural determination. Much of the early appeal of Chlamydomonas was rooted in its promise as a genetic system, but like other classic model organisms, this rise to prominence predated the discovery of the structure of DNA, whole-genome sequences, and molecular techniques for gene manipulation. The haploid genome of C. reinhardtii facilitates genetic analyses and offers many of the advantages of microbial systems applied to a photosynthetic organism. C. reinhardtii has contributed to our understanding of chloroplast-based photosynthesis and cilia biology. Despite pervasive transgene silencing, technological advances have allowed researchers to address outstanding lines of inquiry in algal research. The most thoroughly studied unicellular alga, C. reinhardtii, is the current standard for algal research, and although genome editing is still far from efficient and routine, it nevertheless serves as a template for other algae. We present a historical retrospective of the rise of C. reinhardtii to illuminate its past and present. We also present resources for current and future scientists who may wish to expand their studies to the realm of microalgae.
Assuntos
Chlamydomonas/genética , Chlamydomonas/fisiologia , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/fisiologia , Cloroplastos/genética , Cloroplastos/metabolismo , Edição de Genes , Haploidia , Fotossíntese/genética , Fotossíntese/fisiologiaRESUMO
Light and nutrients are critical regulators of photosynthesis and metabolism in plants and algae. Many algae have the metabolic flexibility to grow photoautotrophically, heterotrophically, or mixotrophically. Here, we describe reversible Glc-dependent repression/activation of oxygenic photosynthesis in the unicellular green alga Chromochloris zofingiensis. We observed rapid and reversible changes in photosynthesis, in the photosynthetic apparatus, in thylakoid ultrastructure, and in energy stores including lipids and starch. Following Glc addition in the light, C. zofingiensis shuts off photosynthesis within days and accumulates large amounts of commercially relevant bioproducts, including triacylglycerols and the high-value nutraceutical ketocarotenoid astaxanthin, while increasing culture biomass. RNA sequencing reveals reversible changes in the transcriptome that form the basis of this metabolic regulation. Functional enrichment analyses show that Glc represses photosynthetic pathways while ketocarotenoid biosynthesis and heterotrophic carbon metabolism are upregulated. Because sugars play fundamental regulatory roles in gene expression, physiology, metabolism, and growth in both plants and animals, we have developed a simple algal model system to investigate conserved eukaryotic sugar responses as well as mechanisms of thylakoid breakdown and biogenesis in chloroplasts. Understanding regulation of photosynthesis and metabolism in algae could enable bioengineering to reroute metabolism toward beneficial bioproducts for energy, food, pharmaceuticals, and human health.
Assuntos
Clorofíceas/fisiologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Glucose/farmacologia , Oxigênio/metabolismo , Fotossíntese/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Antioxidantes/metabolismo , Bioengenharia , Carbono/metabolismo , Clorofíceas/genética , Clorofíceas/efeitos da radiação , Clorofíceas/ultraestrutura , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Fotossíntese/efeitos da radiação , Tilacoides/metabolismo , Tilacoides/ultraestrutura , Transcriptoma/efeitos da radiação , Xantofilas/metabolismoRESUMO
The unicellular green alga Chlamydomonas reinhardtii displays metabolic flexibility in response to a changing environment. We analyzed expression patterns of its three genomes in cells grown under light-dark cycles. Nearly 85% of transcribed genes show differential expression, with different sets of transcripts being up-regulated over the course of the day to coordinate cellular growth before undergoing cell division. Parallel measurements of select metabolites and pigments, physiological parameters, and a subset of proteins allow us to infer metabolic events and to evaluate the impact of the transcriptome on the proteome. Among the findings are the observations that Chlamydomonas exhibits lower respiratory activity at night compared with the day; multiple fermentation pathways, some oxygen-sensitive, are expressed at night in aerated cultures; we propose that the ferredoxin, FDX9, is potentially the electron donor to hydrogenases. The light stress-responsive genes PSBS, LHCSR1, and LHCSR3 show an acute response to lights-on at dawn under abrupt dark-to-light transitions, while LHCSR3 genes also exhibit a later, second burst in expression in the middle of the day dependent on light intensity. Each response to light (acute and sustained) can be selectively activated under specific conditions. Our expression dataset, complemented with coexpression networks and metabolite profiling, should constitute an excellent resource for the algal and plant communities.
Assuntos
Chlamydomonas/genética , Chlamydomonas/metabolismo , Genômica , Metabolômica , Proteômica , Divisão Celular , Replicação do DNA , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genômica/métodos , Glicólise , Metaboloma , Metabolômica/métodos , NAD/metabolismo , Oxirredução , Fotossíntese/genética , Proteoma , Proteômica/métodos , Transdução de Sinais , TranscriptomaRESUMO
Exposing cells to excess metal concentrations well beyond the cellular quota is a powerful tool for understanding the molecular mechanisms of metal homeostasis. Such improved understanding may enable bioengineering of organisms with improved nutrition and bioremediation capacity. We report here that Chlamydomonas reinhardtii can accumulate manganese (Mn) in proportion to extracellular supply, up to 30-fold greater than its typical quota and with remarkable tolerance. As visualized by X-ray fluorescence microscopy and nanoscale secondary ion MS (nanoSIMS), Mn largely co-localizes with phosphorus (P) and calcium (Ca), consistent with the Mn-accumulating site being an acidic vacuole, known as the acidocalcisome. Vacuolar Mn stores are accessible reserves that can be mobilized in Mn-deficient conditions to support algal growth. We noted that Mn accumulation depends on cellular polyphosphate (polyP) content, indicated by 1) a consistent failure of C. reinhardtii vtc1 mutant strains, which are deficient in polyphosphate synthesis, to accumulate Mn and 2) a drastic reduction of the Mn storage capacity in P-deficient cells. Rather surprisingly, X-ray absorption spectroscopy, EPR, and electron nuclear double resonance revealed that only little Mn2+ is stably complexed with polyP, indicating that polyP is not the final Mn ligand. We propose that polyPs are a critical component of Mn accumulation in Chlamydomonas by driving Mn relocation from the cytosol to acidocalcisomes. Within these structures, polyP may, in turn, escort vacuolar Mn to a number of storage ligands, including phosphate and phytate, and other, yet unidentified, compounds.
Assuntos
Chlamydomonas/metabolismo , Íons/metabolismo , Manganês/metabolismo , Vacúolos/efeitos dos fármacos , Cálcio/metabolismo , Chlamydomonas/efeitos dos fármacos , Íons/química , Manganês/toxicidade , Fósforo/metabolismo , Vacúolos/metabolismo , Espectroscopia por Absorção de Raios XRESUMO
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.
Assuntos
Pigmentos Biliares/biossíntese , Chlamydomonas reinhardtii/metabolismo , Heme Oxigenase-1/metabolismo , Proteínas de Plantas/metabolismo , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/efeitos da radiação , Cloroplastos/genética , Cloroplastos/metabolismo , Regulação da Expressão Gênica de Plantas , Heme Oxigenase-1/genética , Luz , Mutação , Oxigênio/metabolismo , Complexo de Proteína do Fotossistema I/genética , Complexo de Proteína do Fotossistema I/metabolismo , Proteínas de Plantas/genética , Transdução de Sinais/genéticaRESUMO
Microalgae have potential to help meet energy and food demands without exacerbating environmental problems. There is interest in the unicellular green alga Chromochloris zofingiensis, because it produces lipids for biofuels and a highly valuable carotenoid nutraceutical, astaxanthin. To advance understanding of its biology and facilitate commercial development, we present a C. zofingiensis chromosome-level nuclear genome, organelle genomes, and transcriptome from diverse growth conditions. The assembly, derived from a combination of short- and long-read sequencing in conjunction with optical mapping, revealed a compact genome of â¼58 Mbp distributed over 19 chromosomes containing 15,274 predicted protein-coding genes. The genome has uniform gene density over chromosomes, low repetitive sequence content (â¼6%), and a high fraction of protein-coding sequence (â¼39%) with relatively long coding exons and few coding introns. Functional annotation of gene models identified orthologous families for the majority (â¼73%) of genes. Synteny analysis uncovered localized but scrambled blocks of genes in putative orthologous relationships with other green algae. Two genes encoding beta-ketolase (BKT), the key enzyme synthesizing astaxanthin, were found in the genome, and both were up-regulated by high light. Isolation and molecular analysis of astaxanthin-deficient mutants showed that BKT1 is required for the production of astaxanthin. Moreover, the transcriptome under high light exposure revealed candidate genes that could be involved in critical yet missing steps of astaxanthin biosynthesis, including ABC transporters, cytochrome P450 enzymes, and an acyltransferase. The high-quality genome and transcriptome provide insight into the green algal lineage and carotenoid production.
Assuntos
Clorófitas/genética , Clorófitas/metabolismo , Genoma de Planta/genética , Microalgas/genética , Sequência de Bases , Biocombustíveis , Mapeamento Cromossômico , Cromossomos de Plantas/genética , Análise de Sequência de DNA , Transcriptoma/genética , Xantofilas/biossíntese , Xantofilas/genéticaRESUMO
Chlamydomonas reinhardtii is a unicellular chlorophyte alga that is widely studied as a reference organism for understanding photosynthesis, sensory and motile cilia, and for development of an algal-based platform for producing biofuels and bio-products. Its highly repetitive, ~205-kbp circular chloroplast genome and ~15.8-kbp linear mitochondrial genome were sequenced prior to the advent of high-throughput sequencing technologies. Here, high coverage shotgun sequencing was used to assemble both organellar genomes de novo. These new genomes correct dozens of errors in the prior genome sequences and annotations. Genome sequencing coverage indicates that each cell contains on average 83 copies of the chloroplast genome and 130 copies of the mitochondrial genome. Using protocols and analyses optimized for organellar transcripts, RNA-Seq was used to quantify their relative abundances across 12 different growth conditions. Forty-six percent of total cellular mRNA is attributable to high expression from a few dozen chloroplast genes. RNA-Seq data were used to guide gene annotation, to demonstrate polycistronic gene expression, and to quantify splicing of psaA and psbA introns. In contrast to a conclusion from a recent study, we found that chloroplast transcripts are not edited. Unexpectedly, cytosine-rich polynucleotide tails were observed at the 3'-end of all mitochondrial transcripts. A comparative genomics analysis of eight laboratory strains and 11 wild isolates of C. reinhardtii identified 2658 variants in the organellar genomes, which is 1/10th as much genetic diversity as is found in the nucleus.
Assuntos
Chlamydomonas reinhardtii/genética , DNA Mitocondrial/genética , Genoma de Cloroplastos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Chlamydomonas reinhardtii/citologia , Edição de Genes , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Genômica/métodos , Anotação de Sequência Molecular , Organelas/genética , Splicing de RNA , Análise de Sequência de RNA/métodosRESUMO
Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) catalyzes the reaction between gaseous carbon dioxide (CO2 ) and ribulose-1,5-bisphosphate. Although it is one of the most studied enzymes, the assembly mechanisms of the large hexadecameric RuBisCO is still emerging. In bacteria and in the C4 plant Zea mays, a protein with distant homology to pterin-4α-carbinolamine dehydratase (PCD) has recently been shown to be involved in RuBisCO assembly. However, studies of the homologous PCD-like protein (RAF2, RuBisCO assembly factor 2) in the C3 plant Arabidopsis thaliana (A. thaliana) have so far focused on its role in hormone and stress signaling. We investigated whether A. thalianaRAF2 is also involved in RuBisCO assembly. We localized RAF2 to the soluble chloroplast stroma and demonstrated that raf2 A. thaliana mutant plants display a severe pale green phenotype with reduced levels of stromal RuBisCO. We concluded that the RAF2 protein is probably involved in RuBisCO assembly in the C3 plant A. thaliana.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , Alelos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Cloroplastos/metabolismo , Técnicas de Inativação de Genes , Filogenia , Alinhamento de Sequência , Tilacoides/metabolismoRESUMO
BACKGROUND: Lichens, encompassing 20,000 known species, are symbioses between specialized fungi (mycobionts), mostly ascomycetes, and unicellular green algae or cyanobacteria (photobionts). Here we describe the first parallel genomic analysis of the mycobiont Cladonia grayi and of its green algal photobiont Asterochloris glomerata. We focus on genes/predicted proteins of potential symbiotic significance, sought by surveying proteins differentially activated during early stages of mycobiont and photobiont interaction in coculture, expanded or contracted protein families, and proteins with differential rates of evolution. RESULTS: A) In coculture, the fungus upregulated small secreted proteins, membrane transport proteins, signal transduction components, extracellular hydrolases and, notably, a ribitol transporter and an ammonium transporter, and the alga activated DNA metabolism, signal transduction, and expression of flagellar components. B) Expanded fungal protein families include heterokaryon incompatibility proteins, polyketide synthases, and a unique set of G-protein α subunit paralogs. Expanded algal protein families include carbohydrate active enzymes and a specific subclass of cytoplasmic carbonic anhydrases. The alga also appears to have acquired by horizontal gene transfer from prokaryotes novel archaeal ATPases and Desiccation-Related Proteins. Expanded in both symbionts are signal transduction components, ankyrin domain proteins and transcription factors involved in chromatin remodeling and stress responses. The fungal transportome is contracted, as are algal nitrate assimilation genes. C) In the mycobiont, slow-evolving proteins were enriched for components involved in protein translation, translocation and sorting. CONCLUSIONS: The surveyed genes affect stress resistance, signaling, genome reprogramming, nutritional and structural interactions. The alga carries many genes likely transferred horizontally through viruses, yet we found no evidence of inter-symbiont gene transfer. The presence in the photobiont of meiosis-specific genes supports the notion that sexual reproduction occurs in Asterochloris while they are free-living, a phenomenon with implications for the adaptability of lichens and the persistent autonomy of the symbionts. The diversity of the genes affecting the symbiosis suggests that lichens evolved by accretion of many scattered regulatory and structural changes rather than through introduction of a few key innovations. This predicts that paths to lichenization were variable in different phyla, which is consistent with the emerging consensus that ascolichens could have had a few independent origins.