Jeanette M. Quinn

The Crd1 gene encodes a putative di-iron enzyme required for photosystem I accumulation in copper deficiency and hypoxia in Chlamydomonas reinhardtii

Chlamydomonas reinhardtii adapts to copper deficiency by degrading apoplastocyanin and inducing Cyc6 and Cpx1 encoding cytochrome c6 and coproporphyrinogen oxidase, respectively. To identify other components in this pathway, colonies resulting from insertional mutagenesis were screened for copper‐ conditional phenotypes. Twelve crd (copper response defect) strains were identified. In copper‐deficient conditions, the crd strains fail to accumulate photosystem I and light‐harvesting complex I, and they contain reduced amounts of light‐harvesting complex II. Cyc6, Cpx1 expression and plastocyanin accumulation remain copper responsive. The crd phenotype is rescued by a similar amount of copper as is required for repression of Cyc6 and Cpx1 and for maintenance of plastocyanin at its usual stoichiometry, suggesting that the affected gene is a target of the same signal transduction pathway. The crd strains represent alleles at a single locus, CRD1, which encodes a 47 kDa, hydrophilic protein with a consensus carboxylate‐bridged di‐iron binding site. Crd1 homologs are present in the genomes of photosynthetic organisms. In Chlamydomonas, Crd1 expression is activated in copper‐ or oxygen‐deficient cells, and Crd1 function is required for adaptation to these conditions.

Copper-dependent iron assimilation pathway in the model photosynthetic eukaryote Chlamydomonas reinhardtii.

ABSTRACT The unicellular green alga Chlamydomonas reinhardtii is a valuable model for studying metal metabolism in a photosynthetic background. A search of the Chlamydomonas expressed sequence tag database led to the identification of several components that form a copper-dependent iron assimilation pathway related to the high-affinity iron uptake pathway defined originally for Saccharomyces cerevisiae. They include a multicopper ferroxidase (encoded by Fox1), an iron permease (encoded by Ftr1), a copper chaperone (encoded by Atx1), and a copper-transporting ATPase. A cDNA, Fer1, encoding ferritin for iron storage also was identified. Expression analysis demonstrated that Fox1 and Ftr1 were coordinately induced by iron deficiency, as were Atx1 and Fer1, although to lesser extents. In addition, Fox1 abundance was regulated at the posttranscriptional level by copper availability. Each component exhibited sequence relationship with its yeast, mammalian, or plant counterparts to various degrees; Atx1 of C. reinhardtii is also functionally related with respect to copper chaperone and antioxidant activities. Fox1 is most highly related to the mammalian homologues hephaestin and ceruloplasmin; its occurrence and pattern of expression in Chlamydomonas indicate, for the first time, a role for copper in iron assimilation in a photosynthetic species. Nevertheless, growth of C. reinhardtii under copper- and iron-limiting conditions showed that, unlike the situation in yeast and mammals, where copper deficiency results in a secondary iron deficiency, copper-deficient Chlamydomonas cells do not exhibit symptoms of iron deficiency. We propose the existence of a copper-independent iron assimilation pathway in this organism.

Oxygen Deficiency Responsive Gene Expression inChlamydomonas reinhardtii through a Copper-Sensing SignalTransduction Pathway

Chlamydomonas reinhardtii activatesCpx1, Cyc6, and Crd1, encoding, respectively, coproporphyrinogen oxidase, cytochromec 6, and a novel di-iron enzyme when transferred to oxygen-deficient growth conditions. This response is physiologically relevant because C. reinhardtiiexperiences these growth conditions routinely, and furthermore, one of the target genes, Crd1, is functionally required for normal growth under oxygen-depleted conditions. The same genes are activated also in response to copper-deficiency through copper-response elements that function as target sites for a transcriptional activator. The core of the copper-response element, GTAC, is required also for the hypoxic response, as is a trans-acting locus, CRR1. Mercuric ions, which antagonize the copper-deficiency response, also antagonize the oxygen-deficiency response of these target genes. Taken together, these observations suggest that the oxygen- and copper-deficiency responses share signal transduction components. Nevertheless, whereas the copper-response element is sufficient for the nutritional copper response, the oxygen-deficiency response requires, in addition, a second cis-element, indicating that the response to oxygen depletion is not identical to the nutritional copper response. The distinction between the two responses is also supported by comparative analysis of the response of the target genes,Cyc6, Cpx1, and Crd1, to copper versus oxygen deficiency. A Crr1-independent pathway forHyd1 expression in oxygen-depleted C. reinhardtii demonstrates the existence of multiple oxygen/redox-responsive circuits in this model organism.

Copper-responsive gene expression during adaptation to copper deficiency.

Publisher Summary This chapter describes the preparation and use of copper-deficient media to study copper-responsive gene expression and copper metabolism with emphasis on Chlamydomonas reinhardtii as an experimental system. The principles discussed in this chapter can also be applied to the study of other trace metal responsive processes. The unicellular green alga C. reinhardtii has proved to be a useful model system for studying one aspect of copper metabolism, specifically, adaptations to copper deficiency. When copper is limiting, certain green algae and cyanobacteria synthesize cytochrome c 6 , which can substitute functionally for plastocyanin. The accumulation of one or the other protein is reciprocally dependent on the presence of copper in the growth medium. In C. reinhardtii , this is affected by enhanced degradation of plastocyanin in copper-deficient cells and activation of transcription of the Cyc6 gene. The extent of transcription of the Cyc6 gene is directly proportional to the perceived copper deficiency. Thus, measurement of Cyc6 expression is generally a good assay for copper deficiency.

Copper response element and Crr1-dependent Ni(2+)-responsive promoter for induced, reversible gene expression in Chlamydomonas reinhardtii.

ABSTRACT The Cpx1 and Cyc6 genes of Chlamydomonas reinhardtii are activated in copper-deficient cells via a signal transduction pathway that requires copper response elements (CuREs) and a copper response regulator defined by the CRR1 locus. The two genes can also be activated by provision of nickel or cobalt ions in the medium. The response to nickel ions requires at least one CuRE and also CRR1 function, suggesting that nickel interferes with a component in the nutritional copper signal transduction pathway. Nickel does not act by preventing copper uptake/utilization because (i) holoplastocyanin formation is unaffected in Ni2+-treated cells and (ii) provision of excess copper cannot reverse the Ni-dependent activation of the target genes. The CuRE is sufficient for conferring Ni-responsive expression to a reporter gene, which suggests that the system has practical application as a vehicle for inducible gene expression. The inducer can be removed either by replacing the medium or by chelating the inducer with excess EDTA, either of which treatments reverses the activation of the target genes.

Induction of coproporphyrinogen oxidase in Chlamydomonas chloroplasts occurs via transcriptional regulation of Cpx1 mediated by copper response elements and increased translation from a copper deficiency-specific form of the transcript.

Coproporphyrinogen III oxidase, encoded by a single nuclear gene in Chlamydomonas reinhardtii, produces three distinct transcripts. One of these transcripts is greatly induced in copper-deficient cells by transcriptional activation, whereas the other forms are copper-insensitive. The induced form of the transcript was expressed coordinately with the cytochromec 6-encoding (Cyc6) gene, which is known to be transcriptionally regulated in copper-deficient cells. The sequence GTAC, which forms the core of a copper response element associated with the Cyc6 gene, is also essential for induction of the Cpx1 gene, suggesting that both are targets of the same signal transduction pathway. The constitutive and induced Cpx1 transcripts have the same half-lives in vivo, and all encode the same polypeptide with a chloroplast-targeting transit sequence, but the shortest one representing the induced form is a 2–4-fold better template for translation than are either of the constitutive forms. The enzyme remains localized to a soluble compartment in the chloroplast even in induced cells, and its abundance is not affected when the tetrapyrrole pathway is manipulated either genetically or by gabaculine treatment.

Molecular Genetic Analysis of Plastocyanin Biosynthesis in Chlamydomonas reinhardtii

Five plastocyanin-deficient mutants were identified from a population of UV-mutagenized Chlamydomonas reinhardtii cells. Genetic complementation experiments indicated that four mutants represented alleles at the PCY1 locus (pcy1-2, pcy1-3, pcy1-4, and pcy1-5). Sequence analysis confirmed that two strains, pcy1-2 and pcy1-3, carry a frameshift (−1) and a nonsense mutation, respectively, while strains pcy1-4 and pcy1-5 synthesize an extended protein as a result of read-through mutations at the stop codon. The C-terminal extension does not affect synthesis or processing of the pre-proteins, but the polypeptides are rapidly degraded after the second (lumenal) processing event. The frameshift mutation in pcy1-2 results in loss of Pcy1 mRNA, as noted previously for strain ac208 (pcy1-1), but the abundance of Pcy1 mRNA in strain pcy1-3, which carries a nonsense mutation at codon 26, is unaffected relative to wild-type cells. The decreased abundance of frameshifted Pcy1 mRNA is attributed to increased degradation rather than decreased synthesis, since the mRNAs can be stabilized by treatment of cells with cycloheximide or anisomycin. The fifth strain has a wild-type plastocyanin-encoding gene, but the strain accumulates apoplastocyanin at the expense of holoplastocyanin. We suggest that the mutation identifies a new locus (PCY2) whose function is required for normal holoplastocyanin accumulation. Like ac208 (pcy1-1), several of the new mutants were suppressed spontaneously owing to accumulation of cytochrome c6 (a functional substitute for plastocyanin). The suppressor mutation(s) displayed Mendelian inheritance and segregated independently from the PCY1 locus, which confirms that regulation of Cyc6 expression is not tightly linked to plastocyanin function.

Evaluation of oxygen response involving differential gene expression in Chlamydomonas reinhardtii.

Publisher Summary This chapter describes the setup of experiments involving the growth of Chlamydomonas cells in liquid cultures at different oxygen concentrations. The unicellular green alga Chlamydomonas has long been used as a model for the study of nutrient-responsive signal transduction, especially in the context of the function of photosynthetic apparatus. It offers experimental advantages for the study of chloroplast function and biogenesis, photosynthesis, flagellar motility and assembly, photoreceptor biochemistry, and sexual mating. Among these are the ability to manipulate the nuclear and also both organellar genomes; facultative photosynthetic growth because of the ability of Chlamydomonas to use acetate for heterotrophic growth; heterothallic mating types that permit classical genetic approaches for the dissection of important biological problems; and considerable genomic information through EST10 and shotgun genome sequencing projects. This chapter discusses Chlamydomonas as a model organism. Connections between oxygen and copper nutrient homeostasis, concepts related to growth media, culturing, bubbling and flask setup, and other culture conditions besides linking hypoxia and copper-deficient responses through the crr1 mutant are also discussed.

Comparative analysis of copper and iron metabolism in photosynthetic eukaryotes vs yeast and mammals

Copper and iron are essential micronutrients for all organisms because of their function as cofactors in enzymes that catalyze redox reactions in fundamental metabolic processes. Prominent examples of such enzymes include cytochrome oxidase in respiration, plastocyanin in photosynthesis, superoxide dismutase in oxidative stress, and ceruloplasmin in iron metabolism. Copper and iron carry out very similar functions in biology because both exhibit stable, redox-interchangeable ionic states with the potential to generate less stable electron-deficient intermediates during multielectron redox reactions involving oxygen chemistry. The major difference between copper and iron in biological systems derives from their individual ligand preferences and coordination geometries. The bioavailability of copper and iron is low so that organisms are faced with the challenge of acquiring sufficient copper and iron for cellular requirements while avoiding the buildup of levels that could lead to cellular toxicity. Over the last decade, it has become apparent that organisms have developed a suite of strategies to combat such challenges, so that an intricate balance between uptake, utilization, storage and detoxification, and efflux pathways for copper and iron exists.

ADAPTATION OF SCENEDESMUS OBLIQUUS (CHLOROPHYCEAE) TO COPPER‐DEFICIENCY: TRANSCRIPTIONAL REGULATION OF PCY1 BUT NOT CPX1

In contrast to the situation in Chlamydomonas reinhardtii, plastocyanin expression in the green alga Scenedesmus obliquus (Turp.) Kütz. (Chlorophyceae) is regulated by copper ion availability at the level of both protein and mRNA accumulation. To study the mechanism of copper‐dependent mRNA accumulation, plastocyanin‐encoding genomic and cDNA clones have been isolated and sequenced. The single nuclear S. obliquus Pcy1 gene contains two introns and a bipartite transit peptide sequence typical of plastid lumen‐localized proteins. Despite the similarity of the S. obliquus and Chlamydomonas reinhardtii Pcy1 genes, the S. obliquus Pcy1 gene was not expressed from its own promoter, nor could the S. obliquus Pcy1 promoter drive expression of a C. reinhardtii gene when it was introduced into the genome of C. reinhardtii cells. Using the cDNA clone as a probe, copper‐responsive regulation of Pcy1 expression was further characterized in S. obliquus. Experiments examining the time course of Pcy1 degradation on depletion of copper argue against a specific mechanism for Pcy1 mRNA decay in S. obliquus, suggesting that the copper‐responsive change in abundance occurs through transcriptional regulation. The Cpx1 gene (encoding coproporphyrinogen III oxidase) also responds differently to changes in cellular copper status in S. obliquus compared to C. reinhardtii. Whereas Cpx1 is highly induced in copper‐deficient cultures of C. reinhardtii, in S. obliquus the Cpx1 gene is not copper responsive.