The Chlamydomonas Genome Project, version 6: Reference assemblies for mating-type plus and minus strains reveal extensive structural mutation in the laboratory.

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.

JGI Plant Gene Atlas: an updateable transcriptome resource to improve functional gene descriptions across the plant kingdom.

Gene functional descriptions offer a crucial line of evidence for candidate genes underlying trait variation. Conversely, plant responses to environmental cues represent important resources to decipher gene function and subsequently provide molecular targets for plant improvement through gene editing. However, biological roles of large proportions of genes across the plant phylogeny are poorly annotated. Here we describe the Joint Genome Institute (JGI) Plant Gene Atlas, an updateable data resource consisting of transcript abundance assays spanning 18 diverse species. To integrate across these diverse genotypes, we analyzed expression profiles, built gene clusters that exhibited tissue/condition specific expression, and tested for transcriptional response to environmental queues. We discovered extensive phylogenetically constrained and condition-specific expression profiles for genes without any previously documented functional annotation. Such conserved expression patterns and tightly co-expressed gene clusters let us assign expression derived additional biological information to 64 495 genes with otherwise unknown functions. The ever-expanding Gene Atlas resource is available at JGI Plant Gene Atlas (https://plantgeneatlas.jgi.doe.gov) and Phytozome (https://phytozome.jgi.doe.gov/), providing bulk access to data and user-specified queries of gene sets. Combined, these web interfaces let users access differentially expressed genes, track orthologs across the Gene Atlas plants, graphically represent co-expressed genes, and visualize gene ontology and pathway enrichments.

Copper economy in Chlamydomonas: prioritized allocation and reallocation of copper to respiration vs. photosynthesis.

Inorganic elements, although required only in trace amounts, permit life and primary productivity because of their functions in catalysis. Every organism has a minimal requirement of each metal based on the intracellular abundance of proteins that use inorganic cofactors, but elemental sparing mechanisms can reduce this quota. A well-studied copper-sparing mechanism that operates in microalgae faced with copper deficiency is the replacement of the abundant copper protein plastocyanin with a heme-containing substitute, cytochrome (Cyt) c6. This switch, which is dependent on a copper-sensing transcription factor, copper response regulator 1 (CRR1), dramatically reduces the copper quota. We show here that in a situation of marginal copper availability, copper is preferentially allocated from plastocyanin, whose function is dispensable, to other more critical copper-dependent enzymes like Cyt oxidase and a ferroxidase. In the absence of an extracellular source, copper allocation to Cyt oxidase includes CRR1-dependent proteolysis of plastocyanin and quantitative recycling of the copper cofactor from plastocyanin to Cyt oxidase. Transcriptome profiling identifies a gene encoding a Zn-metalloprotease, as a candidate effecting copper recycling. One reason for the retention of genes encoding both plastocyanin and Cyt c6 in algal and cyanobacterial genomes might be because plastocyanin provides a competitive advantage in copper-depleted environments as a ready source of copper.

Nitrogen-Sparing Mechanisms in Chlamydomonas Affect the Transcriptome, the Proteome, and Photosynthetic Metabolism.

Nitrogen (N) is a key nutrient that limits global primary productivity; hence, N-use efficiency is of compelling interest in agriculture and aquaculture. We used Chlamydomonas reinhardtii as a reference organism for a multicomponent analysis of the N starvation response. In the presence of acetate, respiratory metabolism is prioritized over photosynthesis; consequently, the N-sparing response targets proteins, pigments, and RNAs involved in photosynthesis and chloroplast function over those involved in respiration. Transcripts and proteins of the Calvin-Benson cycle are reduced in N-deficient cells, resulting in the accumulation of cycle metabolic intermediates. Both cytosolic and chloroplast ribosomes are reduced, but via different mechanisms, reflected by rapid changes in abundance of RNAs encoding chloroplast ribosomal proteins but not cytosolic ones. RNAs encoding transporters and enzymes for metabolizing alternative N sources increase in abundance, as is appropriate for the soil environmental niche of C. reinhardtii. Comparison of the N-replete versus N-deplete proteome indicated that abundant proteins with a high N content are reduced in N-starved cells, while the proteins that are increased have lower than average N contents. This sparing mechanism contributes to a lower cellular N/C ratio and suggests an approach for engineering increased N-use efficiency.

Dynamic Changes in the Transcriptome and Methylome of Chlamydomonas reinhardtii throughout Its Life Cycle.

The green alga Chlamydomonas reinhardtii undergoes gametogenesis and mating upon nitrogen starvation. While the steps involved in its sexual reproductive cycle have been extensively characterized, the genome-wide transcriptional and epigenetic changes underlying different life cycle stages have yet to be fully described. Here, we performed transcriptome and methylome sequencing to quantify expression and DNA methylation from vegetative and gametic cells of each mating type and from zygotes. We identified 361 gametic genes with mating type-specific expression patterns and 627 genes that are specifically induced in zygotes; furthermore, these sex-related gene sets were enriched for secretory pathway and alga-specific genes. We also examined the C. reinhardtii nuclear methylation map with base-level resolution at different life cycle stages. Despite having low global levels of nuclear methylation, we detected 23 hypermethylated loci in gene-poor, repeat-rich regions. We observed mating type-specific differences in chloroplast DNA methylation levels in plus versus minus mating type gametes followed by chloroplast DNA hypermethylation in zygotes. Lastly, we examined the expression of candidate DNA methyltransferases and found three, DMT1a, DMT1b, and DMT4, that are differentially expressed during the life cycle and are candidate DNA methylases. The expression and methylation data we present provide insight into cell type-specific transcriptional and epigenetic programs during key stages of the C. reinhardtii life cycle.

Systems-level analysis of nitrogen starvation-induced modifications of carbon metabolism in a Chlamydomonas reinhardtii starchless mutant.

To understand the molecular basis underlying increased triacylglycerol (TAG) accumulation in starchless (sta) Chlamydomonas reinhardtii mutants, we undertook comparative time-course transcriptomics of strains CC-4348 (sta6 mutant), CC-4349, a cell wall-deficient (cw) strain purported to represent the parental STA6 strain, and three independent STA6 strains generated by complementation of sta6 (CC-4565/STA6-C2, CC-4566/STA6-C4, and CC-4567/STA6-C6) in the context of N deprivation. Despite N starvation-induced dramatic remodeling of the transcriptome, there were relatively few differences (5 × 10(2)) observed between sta6 and STA6, the most dramatic of which were increased abundance of transcripts encoding key regulated or rate-limiting steps in central carbon metabolism, specifically isocitrate lyase, malate synthase, transaldolase, fructose bisphosphatase and phosphoenolpyruvate carboxykinase (encoded by ICL1, MAS1, TAL1, FBP1, and PCK1 respectively), suggestive of increased carbon movement toward hexose-phosphate in sta6 by upregulation of the glyoxylate pathway and gluconeogenesis. Enzyme assays validated the increase in isocitrate lyase and malate synthase activities. Targeted metabolite analysis indicated increased succinate, malate, and Glc-6-P and decreased Fru-1,6-bisphosphate, illustrating the effect of these changes. Comparisons of independent data sets in multiple strains allowed the delineation of a sequence of events in the global N starvation response in C. reinhardtii, starting within minutes with the upregulation of alternative N assimilation routes and carbohydrate synthesis and subsequently a more gradual upregulation of genes encoding enzymes of TAG synthesis. Finally, genome resequencing analysis indicated that (1) the deletion in sta6 extends into the neighboring gene encoding respiratory burst oxidase, and (2) a commonly used STA6 strain (CC-4349) as well as the sequenced reference (CC-503) are not congenic with respect to sta6 (CC-4348), underscoring the importance of using complemented strains for more rigorous assignment of phenotype to genotype.

Subcellular metal imaging identifies dynamic sites of Cu accumulation in Chlamydomonas.

We identified a Cu-accumulating structure with a dynamic role in intracellular Cu homeostasis. During Zn limitation, Chlamydomonas reinhardtii hyperaccumulates Cu, a process dependent on the nutritional Cu sensor CRR1, but it is functionally Cu deficient. Visualization of intracellular Cu revealed major Cu accumulation sites coincident with electron-dense structures that stained positive for low pH and polyphosphate, suggesting that they are lysosome-related organelles. Nano-secondary ion MS showed colocalization of Ca and Cu, and X-ray absorption spectroscopy was consistent with Cu(+) accumulation in an ordered structure. Zn resupply restored Cu homeostasis concomitant with reduced abundance of these structures. Cu isotope labeling demonstrated that sequestered Cu(+) became bioavailable for the synthesis of plastocyanin, and transcriptome profiling indicated that mobilized Cu became visible to CRR1. Cu trafficking to intracellular accumulation sites may be a strategy for preventing protein mismetallation during Zn deficiency and enabling efficient cuproprotein metallation or remetallation upon Zn resupply.

Fe sparing and Fe recycling contribute to increased superoxide dismutase capacity in iron-starved Chlamydomonas reinhardtii.

Fe deficiency is one of several abiotic stresses that impacts plant metabolism because of the loss of function of Fe-containing enzymes in chloroplasts and mitochondria, including cytochromes, FeS proteins, and Fe superoxide dismutase (FeSOD). Two pathways increase the capacity of the Chlamydomonas reinhardtii chloroplast to detoxify superoxide during Fe limitation stress. In one pathway, MSD3 is upregulated at the transcriptional level up to 10(3)-fold in response to Fe limitation, leading to synthesis of a previously undiscovered plastid-specific MnSOD whose identity we validated immunochemically. In a second pathway, the plastid FeSOD is preferentially retained over other abundant Fe proteins, heme-containing cytochrome f, diiron magnesium protoporphyrin monomethyl ester cyclase, and Fe2S2-containing ferredoxin, demonstrating prioritized allocation of Fe within the chloroplast. Maintenance of FeSOD occurs, after an initial phase of degradation, by de novo resynthesis in the absence of extracellular Fe, suggesting the operation of salvage mechanisms for intracellular recycling and reallocation.

Zinc deficiency impacts CO2 assimilation and disrupts copper homeostasis in Chlamydomonas reinhardtii.

Zinc is an essential nutrient because of its role in catalysis and in protein stabilization, but excess zinc is deleterious. We distinguished four nutritional zinc states in the alga Chlamydomonas reinhardtii: toxic, replete, deficient, and limited. Growth is inhibited in zinc-limited and zinc-toxic cells relative to zinc-replete cells, whereas zinc deficiency is visually asymptomatic but distinguished by the accumulation of transcripts encoding ZIP family transporters. To identify targets of zinc deficiency and mechanisms of zinc acclimation, we used RNA-seq to probe zinc nutrition-responsive changes in gene expression. We identified genes encoding zinc-handling components, including ZIP family transporters and candidate chaperones. Additionally, we noted an impact on two other regulatory pathways, the carbon-concentrating mechanism (CCM) and the nutritional copper regulon. Targets of transcription factor Ccm1 and various CAH genes are up-regulated in zinc deficiency, probably due to reduced carbonic anhydrase activity, validated by quantitative proteomics and immunoblot analysis of Cah1, Cah3, and Cah4. Chlamydomonas is therefore not able to grow photoautotrophically in zinc-limiting conditions, but supplementation with 1% CO2 restores growth to wild-type rates, suggesting that the inability to maintain CCM is a major consequence of zinc limitation. The Crr1 regulon responds to copper limitation and is turned on in zinc deficiency, and Crr1 is required for growth in zinc-limiting conditions. Zinc-deficient cells are functionally copper-deficient, although they hyperaccumulate copper up to 50-fold over normal levels. We suggest that zinc-deficient cells sequester copper in a biounavailable form, perhaps to prevent mismetallation of critical zinc sites.

Remodeling of membrane lipids in iron-starved Chlamydomonas.

Chlamydomonas reinhardtii cells exposed to abiotic stresses (e.g. nitrogen, zinc, or phosphorus deficiency) accumulate triacylglycerols (TAG), which are stored in lipid droplets. Here, we report that iron starvation leads to formation of lipid droplets and accumulation of TAGs. This occurs between 12 and 24 h after the switch to iron-starvation medium. C. reinhardtii cells deprived of iron have more saturated fatty acid (FA), possibly due to the loss of function of FA desaturases, which are iron-requiring enzymes with diiron centers. The abundance of a plastid acyl-ACP desaturase (FAB2) is decreased to the same degree as ferredoxin. Ferredoxin is a substrate of the desaturases and has been previously shown to be a major target of the iron deficiency response. The increase in saturated FA (C16:0 and C18:0) is concomitant with the decrease in unsaturated FA (C16:4, C18:3, or C18:4). This change was gradual for diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) and digalactosyldiacylglycerol (DGDG), whereas the monogalactosyldiacylglycerol (MGDG) FA profile remained stable during the first 12 h, whereas MGDG levels were decreasing over the same period of time. These changes were detectable after only 2 h of iron starvation. On the other hand, DGTS and DGDG contents gradually decreased until a minimum was reached after 24-48 h. RNA-Seq analysis of iron-starved C. reinhardtii cells revealed notable changes in many transcripts coding for enzymes involved in FA metabolism. The mRNA abundances of genes coding for components involved in TAG accumulation (diacylglycerol acyltransferases or major lipid droplet protein) were increased. A more dramatic increase at the transcript level has been observed for many lipases, suggesting that major remodeling of lipid membranes occurs during iron starvation in C. reinhardtii.

Systems biology approach in Chlamydomonas reveals connections between copper nutrition and multiple metabolic steps.

In this work, we query the Chlamydomonas reinhardtii copper regulon at a whole-genome level. Our RNA-Seq data simulation and analysis pipeline validated a 2-fold cutoff and 10 RPKM (reads per kilobase of mappable length per million mapped reads) (~1 mRNA per cell) to reveal 63 CRR1 targets plus another 86 copper-responsive genes. Proteomic and immunoblot analyses captured 25% of the corresponding proteins, whose abundance was also dependent on copper nutrition, validating transcriptional regulation as a major control mechanism for copper signaling in Chlamydomonas. The impact of copper deficiency on the expression of several O2-dependent enzymes included steps in lipid modification pathways. Quantitative lipid profiles indicated increased polyunsaturation of fatty acids on thylakoid membrane digalactosyldiglycerides, indicating a global impact of copper deficiency on the photosynthetic apparatus. Discovery of a putative plastid copper chaperone and a membrane protease in the thylakoid suggest a mechanism for blocking copper utilization in the chloroplast. We also found an example of copper sparing in the N assimilation pathway: the replacement of copper amine oxidase by a flavin-dependent backup enzyme. Forty percent of the targets are previously uncharacterized proteins, indicating considerable potential for new discovery in the biology of copper.

Three acyltransferases and nitrogen-responsive regulator are implicated in nitrogen starvation-induced triacylglycerol accumulation in Chlamydomonas.

Algae have recently gained attention as a potential source for biodiesel; however, much is still unknown about the biological triggers that cause the production of triacylglycerols. We used RNA-Seq as a tool for discovering genes responsible for triacylglycerol (TAG) production in Chlamydomonas and for the regulatory components that activate the pathway. Three genes encoding acyltransferases, DGAT1, DGTT1, and PDAT1, are induced by nitrogen starvation and are likely to have a role in TAG accumulation based on their patterns of expression. DGAT1 and DGTT1 also show increased mRNA abundance in other TAG-accumulating conditions (minus sulfur, minus phosphorus, minus zinc, and minus iron). Insertional mutants, pdat1-1 and pdat1-2, accumulate 25% less TAG compared with the parent strain, CC-4425, which demonstrates the relevance of the trans-acylation pathway in Chlamydomonas. The biochemical functions of DGTT1 and PDAT1 were validated by rescue of oleic acid sensitivity and restoration of TAG accumulation in a yeast strain lacking all acyltransferase activity. Time course analyses suggest than a SQUAMOSA promoter-binding protein domain transcription factor, whose mRNA increases precede that of lipid biosynthesis genes like DGAT1, is a candidate regulator of the nitrogen deficiency responses. An insertional mutant, nrr1-1, accumulates only 50% of the TAG compared with the parental strain in nitrogen-starvation conditions and is unaffected by other nutrient stresses, suggesting the specificity of this regulator for nitrogen-deprivation conditions.

The CRR1 nutritional copper sensor in Chlamydomonas contains two distinct metal-responsive domains.

Copper response regulator 1 (CRR1), an SBP-domain transcription factor, is a global regulator of nutritional copper signaling in Chlamydomonas reinhardtii and activates genes necessary during periods of copper deficiency. We localized Chlamydomonas CRR1 to the nucleus in mustard (Sinapis alba) seedlings, a location consistent with its function as a transcription factor. The Zn binding SBP domain of CRR1 binds copper ions in vitro. Cu(I) can replace Zn(II), but the Cu(II) form is unstable. The DNA binding activity is inhibited in vitro by Cu(II) or Hg(II) ions, which also prevent activation of transcription in vivo, but not by Co(II) or Ni(II), which have no effect in vivo. Copper inhibition of DNA binding is reduced by mutation of a conserved His residue. These results implicate the SBP domain in copper sensing. Deletion of a C-terminal metallothionein-like Cys-rich domain impacted neither nutritional copper signaling nor the effect of mercuric supplementation, but rendered CRR1 insensitive to hypoxia and to nickel supplementation, which normally activate the copper deficiency regulon in wild-type cells. Strains carrying the crr1-ΔCys allele upregulate ZRT genes and hyperaccumulate Zn(II), suggesting that the effect of nickel ions may be revealing a role for the C-terminal domain of CRR1 in zinc homeostasis in Chlamydomonas.

A revised mineral nutrient supplement increases biomass and growth rate in Chlamydomonas reinhardtii.

Interest in exploiting algae as a biofuel source and the role of inorganic nutrient deficiency in inducing triacylglyceride (TAG) accumulation in cells necessitates a strategy to efficiently formulate species-specific culture media that can easily be manipulated. Using the reference organism Chlamydomonas reinhardtii, we tested the hypothesis that modeling trace element supplements after the cellular ionome would result in optimized cell growth. We determined the trace metal content of several commonly used Chlamydomonas strains in various culture conditions and developed a revised trace element solution to parallel these measurements. Comparison of cells growing in the revised supplement versus a traditional trace element solution revealed faster growth rates and higher maximum cell densities with the revised recipe. RNA-seq analysis of cultures growing in the traditional versus revised medium suggest that the variation in transcriptomes was smaller than that found between different wild-type strains grown in traditional Hutner's supplement. Visual observation did not reveal defects in cell motility or mating efficiency in the new supplement. Ni²âº-inducible expression from the CYC6 promoter remained a useful tool, albeit with an increased requirement for Ni²âº because of the introduction of an EDTA buffer system in the revised medium. Other advantages include more facile preparation of trace element stock solutions, a reduction in total chemical use, a more consistent batch-to-batch formulation and long-term stability (tested up to 5 years). Under the new growth regime, we analyzed cells growing under different macro- and micronutrient deficiencies. TAG accumulation in N deficiency is comparable in the new medium. Fe and Zn deficiency also induced TAG accumulation, as suggested by Nile Red staining. This approach can be used to efficiently optimize culture conditions for other algal species to improve growth and to assay cell physiology.

CCS5, a thioredoxin-like protein involved in the assembly of plastid c-type cytochromes.

The c-type cytochromes are metalloproteins with a heme molecule covalently linked to the sulfhydryls of a CXXCH heme-binding site. In plastids, at least six assembly factors are required for heme attachment to the apo-forms of cytochrome f and cytochrome c(6) in the thylakoid lumen. CCS5, controlling plastid cytochrome c assembly, was identified through insertional mutagenesis in the unicellular green alga Chlamydomonas reinhardtii. The complementing gene encodes a protein with similarity to Arabidopsis thaliana HCF164, which is a thylakoid membrane-anchored protein with a lumen-facing thioredoxin-like domain. HCF164 is required for cytochrome b(6)f biogenesis, but its activity and site of action in the assembly process has so far remained undeciphered. We show that CCS5 is a component of a trans-thylakoid redox pathway and operates by reducing the CXXCH heme-binding site of apocytochrome c prior to the heme ligation reaction. The proposal is based on the following findings: 1) the ccs5 mutant is rescued by exogenous thiols; 2) CCS5 interacts with apocytochrome f and c(6) in a yeast two-hybrid assay; and 3) recombinant CCS5 is able to reduce a disulfide in the CXXCH heme-binding site of apocytochrome f.

Zinc binding drives sheet formation by the SAM domain of diacylglycerol kinase δ.

The diacylglycerol kinase (DGK) family of enzymes plays critical roles in lipid signaling pathways by converting diacylglycerol to phosphatidic acid, thereby downregulating signaling by the former and upregulating signaling by the latter second messenger. Ten DGK family isozymes have been identified to date, which possess different interaction motifs imparting distinct temporal and spatial control of DGK activity to each isozyme. Two DGK family members, δ and η, contain a sterile alpha motif (SAM) domain. The SAM domain of DGKδ1 forms helical polymers that are important for retaining the enzyme in cytoplasmic puncta, thereby inhibiting activity at the plasma membrane until pathway activation. Because zinc was found to be important for stabilizing the similar SAM polymers of the scaffolding protein Shank-3, we investigated the potential role of zinc in DGKδ SAM domain (DGKδSAM) assembly. We find that DGKδSAM binds zinc at multiple sites, driving the organization of the DGKδSAM into large sheets of polymers. Moreover, a mutant DGKδ containing a SAM domain refractory to zinc binding diminishes the formation of cytoplasmic puncta, shows partially impaired regulation of transport to the plasma membrane, and lacks the ability to inhibit the formation of CopII coated vesicles. These results suggest that zinc may play an important role in the assembly and physiology of the DGKδ isozyme.

A subset of the diverse COG0523 family of putative metal chaperones is linked to zinc homeostasis in all kingdoms of life.

BACKGROUND: COG0523 proteins are, like the nickel chaperones of the UreG family, part of the G3E family of GTPases linking them to metallocenter biosynthesis. Even though the first COG0523-encoding gene, cobW, was identified almost 20 years ago, little is known concerning the function of other members belonging to this ubiquitous family. RESULTS: Based on a combination of comparative genomics, literature and phylogenetic analyses and experimental validations, the COG0523 family can be separated into at least fifteen subgroups. The CobW subgroup involved in cobalamin synthesis represents only one small sub-fraction of the family. Another, larger subgroup, is suggested to play a predominant role in the response to zinc limitation based on the presence of the corresponding COG0523-encoding genes downstream from putative Zur binding sites in many bacterial genomes. Zur binding sites in these genomes are also associated with candidate zinc-independent paralogs of zinc-dependent enzymes. Finally, the potential role of COG0523 in zinc homeostasis is not limited to Bacteria. We have predicted a link between COG0523 and regulation by zinc in Archaea and show that two COG0523 genes are induced upon zinc depletion in a eukaryotic reference organism, Chlamydomonas reinhardtii. CONCLUSION: This work lays the foundation for the pursuit by experimental methods of the specific role of COG0523 members in metal trafficking. Based on phylogeny and comparative genomics, both the metal specificity and the protein target(s) might vary from one COG0523 subgroup to another. Additionally, Zur-dependent expression of COG0523 and putative paralogs of zinc-dependent proteins may represent a mechanism for hierarchal zinc distribution and zinc sparing in the face of inadequate zinc nutrition.

A ferroxidase encoded by FOX1 contributes to iron assimilation under conditions of poor iron nutrition in Chlamydomonas.

When the abundance of the FOX1 gene product is reduced, Chlamydomonas cells grow poorly in iron-deficient medium, but not in iron-replete medium, suggesting that FOX1-dependent iron uptake is a high-affinity pathway. Alternative pathways for iron assimilation, such as those involving ZIP family transporters IRT1 and IRT2, may be operational.

Regulation and localization of isoforms of the aerobic oxidative cyclase in Chlamydomonas reinhardtii.

Tetrapyrrole synthesis is complex and is regulated at several points. The aerobic oxidative cyclase catalyzes the oxidative closure of the fifth ring characteristic of all chlorophylls. Chlamydomonas reinhardtii encodes two paralogous protein components of the cyclase, which are differentially accumulated based on copper nutrition and oxygen supply. CRD1 is accumulated in copper-deficient conditions, whereas CTH1 is present in copper replete conditions. Here, we show that CRD1 expression is regulated through a copper-responsive element located upstream of the transcription start site. The differential production of CRD1 transcript accounts for the differential accumulation of the corresponding polypeptide. The CTH1 locus produces two transcripts: a 2.1 kb one under copper-replete conditions and a 3.1 kb one under copper-deficient conditions. We show that the 2.1 kb transcript can be translated into protein in vitro whereas the 3.1 kb transcript cannot. Differential accumulation of the 2.1 vs the 3.1 kb transcript therefore accounts for the copper-responsive accumulation of CTH1. Biochemical fractionation reveals that both CRD1 and CTH1 are localized to chloroplast envelope as well as thylakoid membranes.