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

Abstract

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.

Oxygen Deficiency Responsive Gene Expression in Chlamydomonas reinhardtii through a Copper-Sensing Signal Transduction Pathway

“…Each is activated during adaptation to copper deficiency. We noted that these three genes are also induced in copper-replete cells under oxygen-deficient or near anoxic conditions as might be experienced in their natural environment or during normal laboratory growth conditions (Moseley et al, 2000;Quinn et al, 2000a). For instance, when heterotrophic C. reinhardtii cultures are suspended by slow basal stirring (versus vigorous agitation on a shaker at 250 rpm) under normal room lighting (approximately 10-15 mol m Ϫ2 s Ϫ1 ), they can become oxygen depleted within a day as a consequence of respiratory activity (Fig.…”

“…Therefore, copper-deficient crd1 strains are chlorotic (Moseley et al, 2000). If expression of Crd1 in hypoxic cells represents a requirement for its gene product in hypoxic cells, crd1 mutants should display chlorosis under hypoxic growth conditions.…”

“…Increased expression of Crd1 might be explained on the same basis. Crd1 is proposed to catalyze an oxygendependent oxidation in an Fe deficiency-sensitive pathway for cofactor biosynthesis in the plastid (Moseley et al, 2000;Pinta et al, 2002). Perhaps Crd1 becomes rate limiting for this pathway in oxygendeficient cells.…”

“…Cytochrome c 6 is induced in copper-deficiency by transcriptional activation of the Cyc6 gene through copper-response elements (CuREs). C. reinhardtii also up-regulates Cpx1 (encoding coprogen oxidase, an enzyme of tetrapyrrole biosynthesis) and Crd1 (encoding a putative di-iron enzyme) during adaptation to copper deficiency (Hill and Merchant, 1995;Moseley et al, 2000;Quinn et al, 2000a). Because each copper-responsive gene is also expressed in hypoxic cells, a mechanistic and physiological connection between copper-and oxygendeficiency-induced gene expression was proposed (Moseley et al, 2000;Quinn et al, 2000a).…”

“…C. reinhardtii also up-regulates Cpx1 (encoding coprogen oxidase, an enzyme of tetrapyrrole biosynthesis) and Crd1 (encoding a putative di-iron enzyme) during adaptation to copper deficiency (Hill and Merchant, 1995;Moseley et al, 2000;Quinn et al, 2000a). Because each copper-responsive gene is also expressed in hypoxic cells, a mechanistic and physiological connection between copper-and oxygendeficiency-induced gene expression was proposed (Moseley et al, 2000;Quinn et al, 2000a). The availability of well-characterized target genes and the amenability of C. reinhardtii for molecular genetic analysis of signaling pathways indicated that oxygen deficiency-activated expression of Cyc6, Cpx1, and Crd1 might serve as another important model for hypoxic responses in photosynthetic eukaryotes, particularly in the context of chloroplast metabolism.…”