Genetic dissection of nutritional copper signaling in chlamydomonas distinguishes regulatory and target genes

Abstract

A genetic screen for Chlamydomonas reinhardtii mutants with copper-dependent growth or nonphotosynthetic phenotypes revealed three loci, COPPER RESPONSE REGULATOR 1 (CRR1), COPPER RESPONSE DEFECT 1 (CRD1), and COPPER RESPONSE DEFECT 2 (CRD2), distinguished as regulatory or target genes on the basis of phenotype. CRR1 was shown previously to be required for transcriptional activation of target genes like CYC6, CPX1, and CRD1, encoding, respectively, cytochrome c(6) (which is a heme-containing substitute for copper-containing plastocyanin), coproporphyrinogen III oxidase, and Mg-protoporphyrin IX monomethylester cyclase. We show here that CRR1 is required also for normal accumulation of copper proteins like plastocyanin and ferroxidase in copper-replete medium and for apoplastocyanin degradation in copper-deficient medium, indicating that a single pathway controls nutritional copper homeostasis at multiple levels. CRR1 is linked to the SUPPRESSOR OF PCY1-AC208 13 (SOP13) locus, which corresponds to a gain-of-function mutation resulting in copper-independent expression of CYC6. CRR1 is required also for hypoxic growth, pointing to a physiologically meaningful regulatory connection between copper deficiency and hypoxia. The growth phenotype of crr1 strains results primarily from secondary iron deficiency owing to reduced ferroxidase abundance, suggesting a role for CRR1 in copper distribution to a multicopper ferroxidase involved in iron assimilation. Mutations at the CRD2 locus also result in copper-conditional iron deficiency, which is consistent with a function for CRD2 in a pathway for copper delivery to the ferroxidase. Taken together, the observations argue for a specialized copper-deficiency adaptation for iron uptake in Chlamydomonas.

Figure 1.—

Growth phenotypes of candidate mutants. (A) Appearance of cultures after growth for 5 days [wild type (CC125), crr1-1, and crd2-1] or 6 days (crd1-5), in TAP medium supplemented with the indicated Cu concentrations. (B) Appearance of the same wild-type, crr1-1, and crd2-1 cultures after 13 days of growth. (C) Plot of cell density over time for wild-type (CC125), crr1-1, crd1-5, and crd2-1 cultures over 12 days of growth in TAP medium supplemented with 6 μm (+) or 0 μm (−) Cu. (D) Chlorophyll fluorescence induction curves from wild-type vs. mutant strains grown on solid TAP medium, with (+, 6 μm) or without (−, 0 μm) Cu.

Figure 1.—

Growth phenotypes of candidate mutants. (A) Appearance of cultures after growth for 5 days [wild type (CC125), crr1-1, and crd2-1] or 6 days (crd1-5), in TAP medium supplemented with the indicated Cu concentrations. (B) Appearance of the same wild-type, crr1-1, and crd2-1 cultures after 13 days of growth. (C) Plot of cell density over time for wild-type (CC125), crr1-1, crd1-5, and crd2-1 cultures over 12 days of growth in TAP medium supplemented with 6 μm (+) or 0 μm (−) Cu. (D) Chlorophyll fluorescence induction curves from wild-type vs. mutant strains grown on solid TAP medium, with (+, 6 μm) or without (−, 0 μm) Cu.

Figure 2.—

Biochemical phenotypes of crr and crd strains. (A) RNA blot analysis comparing expression of Cu-responsive genes in wild-type vs. mutant cells. Three micrograms of total RNA was loaded in each lane. RNA was prepared from cultures grown in TAP medium supplemented with 6 μm (+) or 0 μm (−) Cu. −Cu crr1-1 and crr1-2 cultures were transferred to Cu-free medium once; all other −Cu cultures were transferred at least twice. CYC6 encodes cyt c₆; CRD1 encodes a component of the protoporphyrin IX monomethylester cyclase, formerly known as the COPPER RESPONSE DEFECT 1 gene; and CBLP encodes a Chlamydomonas G-protein β-subunit-like protein, used here as a loading control. (B) Immunoblots to compare accumulation of Cu-responsive proteins in wild-type vs. mutant cells. The migration positions of apo-, di-apo-, and holoforms of plastocyanin are indicated with arrows in the first panel, as is the position of cyt c₆, which cross-reacts with the anti-plastocyanin antibody. In the fourth panel, arrows indicate the positions of the Cth1 and Crd1 gene products, which are both recognized by the anti-Crd1 antiserum. (C) Immunoblot showing the accumulation of cyt c₆, apo-, di-apo-, and holoplastocyanin in wild-type cells compared to crr1-1 cells grown in TAP medium with the indicated concentrations (micromolar) of Cu.

Figure 3.—

Acclimation responses to Fe deficiency in wild-type vs. crr1 strains. (A) Appearance of wild-type and crr1-1 cultures grown in +Fe (18 μm) and +Cu (6 μm), washed with −Fe −Cu TAP and grown for 3 days in 1 μm Fe TAP with or without 6 μm added Cu, as indicated. crr1-1 cells grown without copper contained ∼50% of wild-type chlorophyll. (B) RNA blot analysis comparing FOX1 mRNA expression in CC-125 (wild type) and crr1-1 mutant strains. CC-125 or crr1-1 cells were grown in TAP medium containing the indicated concentrations of Fe and Cu. Five micrograms of total RNA was loaded per lane. (C) Immunoblots demonstrating accumulation of ferroxidase in wild-type and crr1-1 cells, grown in TAP medium with the indicated concentrations of Fe and Cu. (D) Appearance of crr1-1 cultures grown for 6 days in TAP medium with the indicated concentrations of Fe and Cu, demonstrating partial rescue of the −Cu growth phenotype with excess Fe.

Figure 4.—

Hypoxic growth of wild type vs. the crr1 strain. (A) Wild-type (CC-125) and crr1-1 strains were grown in TAP medium with 6 μm Cu (blue lines) or without Cu (red lines), bubbled with gas mixtures containing the indicated amounts of air (99.8% vs. 1.8%). All mixtures contained 0.2% CO₂, and the balance of the 1.8% air mixture was made up with N₂ (98% N₂). (B) Immunoblot analysis of plastocyanin and cyt c₆ accumulation in soluble protein fractions from wild-type and crr1-1 strains grown for 4 days in 6 μm Cu with the indicated amounts of air. Detection of OEE1 was used to demonstrate equal loading.

Figure 5.—

Acclimation responses to Fe deficiency in wild-type vs. crd2 strains. (A) Growth of crd2-1 vs. wild type in +Cu (6 μm) and −Cu (0 μm) TAP with the indicated amounts of added Fe. (B) RNA blot analysis of genes involved in Fe uptake and distribution in wild type and crd2-1 grown in TAP medium with normal Fe supplementation (18 μm) and with the indicated concentration of added Cu. The FEA1 panel and the bottom CBLP panel are separated from the other panels by a line to indicate that these RNA blots were performed with different RNA samples. Three micrograms of total RNA was loaded per lane. CBLP expression is used to demonstrate equal loading of the samples, and CYC6 expression is used as a marker for copper deficiency. (C) Quantitative RT-PCR analysis of the samples described in B. Ten microliters of product was removed from the amplification reaction and analyzed by electrophoresis to authenticate the product on the basis of size and to visualize the difference in the abundance of the specific mRNA in the input RNA sample. Samples were removed at the following cycles: CTP1, 27; CTP2, 27; ATX1, 30; and CBLP, 19. For the experiment shown, CTP1 is twofold induced in the low copper crd2 samples relative to the saturating copper sample. (D) Immunoblot analysis of ferroxidase accumulation in wild-type and crd2-1 cells grown in +Fe (18 μm) and +Cu (6 μm), washed with −Fe −Cu TAP, and grown for 24 hr in 1 μm Fe TAP with or without 6 μm added Cu, as indicated. The position of the ∼140-kD ferroxidase protein is indicated with an arrow. Higher mobility bands, which are stoichiometrically more prevalent in −Cu wild-type cells or in crd2 mutant cells, are likely to be degradation products. Degradation may be more pronounced under these conditions because of a different conformation of the protein when the copper sites are not fully loaded (Hellman et al. 2002).

Figure 6.—

Biochemical and genetic characterization of a pcy1-ac208 suppressor strain. (A) Immunoblot analysis of soluble extracts to compare expression of plastocyanin and cyt c₆ in wild type, pcy1-ac208, and a pcy1-ac208 suppressor strain, (pcy1-ac208sop13), grown in TAP medium supplemented with the indicated concentrations of Cu (micromolars). Dilution series of wild-type extracts enables estimation of plastocyanin and cyt c₆ abundance. Samples corresponding to equal amounts of chlorophyll were loaded in each lane, and immunodetection of OEE1 was used to demonstrate equal loading. (B) Anti-plastocyanin and cyt c₆ immunoblots of +Cu whole cell extracts illustrating phenotypic analysis of two representative tetrads from a cross of crr1-1 and pcy1-ac208sop13. Cyt c₆ expression in +Cu cells indicates the presence of the sop13 mutation. Accumulation of cyt c₆ in two vegetative diploid strains that are heterozygous for sop13 is also shown.

Figure 7.—

Photosynthesis phenotypes of crr1 and sop13 strains. Shown are fluorescence rise and decay kinetics of +Cu cells from strains B1–B4 (shown in Figure 6B). B1, CRR1pcy1-ac208sop13; B2, crr1-1PCY1SOP13; B3, crr1-1PCY1SOP13; and B4, CRR1pcy1-ac208sop13.