Oxygen deficiency responsive gene expression in Chlamydomonas reinhardtii through a copper-sensing signal transduction pathway

Abstract

Chlamydomonas reinhardtii activates Cpx1, Cyc6, and Crd1, encoding, respectively, coproporphyrinogen oxidase, cytochrome c(6), and a novel di-iron enzyme when transferred to oxygen-deficient growth conditions. This response is physiologically relevant because C. reinhardtii experiences 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 for Hyd1 expression in oxygen-depleted C. reinhardtii demonstrates the existence of multiple oxygen/redox-responsive circuits in this model organism.

Figure 1

A, Cyc6 expression in copper-supplemented laboratory cultures of wild-type C. reinhardtii as a consequence of oxygen depletion. Cells were grown in Erlenmeyer flasks fitted with cotton plugs and were suspended by low basal stirring using a magnetic stir bar and stir plate under normal room lighting (approximately 1–15 μmol m⁻² s⁻¹ illumination). The oxygen content of the culture was measured with a standardized oxygen electrode each day (□). The culture was sampled at the same time for preparation of RNA and was analyzed by hybridization (▵). B, Hypoxia-induced gene expression in C. reinhardtii. Wild-type strain CC125 was grown in copper-supplemented (6 μm) Tris-acetate-phosphate medium under normal aeration to a concentration of 1 × 10⁶ cells mL⁻¹, and it was then bubbled with a gas mixture containing the indicated amount of air plus 2% CO₂ with the balance as N₂. Total RNA was prepared after 24 h and was analyzed by hybridization.

Figure 2

Time course of the Cyc6 and Cpx1 responses. A, Cultures were bubbled with 98% N₂/2% CO₂, sampled in duplicate at the indicated times, and analyzed for RNA abundance by blot hybridization. B, In a comparable time course experiment, cultures were sampled and analyzed for protein accumulation by immunoblotting. The lanes marked −Cu represent the accumulation of coprogen oxidase and cytochrome c₆ in copper-deficient, fully aerated cultures.

Figure 3

Intracellular copper availability in hypoxic-treated cells. Duplicate copper-deficient cultures of wild-type cells were kept in air or transferred to 2% air (CO₂ was kept constant at 2%, balance N₂) for 1 h prior to addition of copper to 6 μm final concentration (t = 0). Soluble protein was prepared from an aliquot of each culture at the indicated times after addition of copper and was analyzed by native gel electrophoresis and immunoblotting using anti-plastocyanin antiserum at 1:1,000 dilution. The antiserum, generated against plastocyanin, cross-reacts with cytochrome c₆ and, therefore, both proteins are detected (lanes 1 and 2).

Figure 4

Effect of oxygen-deficient growth on a crd1 mutant strain. Strain crd1 and wild-type (WT) cells were grown under normal aeration to 1 × 10⁶ cells mL⁻¹. Each culture was then bubbled with a mixture of 2% air (constant 2% CO₂, balance N₂) or with 100% air, and was grown continuously under these conditions for about two generations.

Figure 5

Effect of copper and oxygen deficiency on Cpx1 and Cyc6 expression. Total RNA isolated from cultures grown under the indicated conditions was analyzed. −O₂, 1% air (constant 2% CO₂, balance N₂).

Figure 6

Oxygen deficiency expression of Cpx1-Ars2 reporter gene constructs. Strains containing the indicated Cpx1 5′-upstream sequences fused to the Ars2 reporter gene were grown to 2 × 10⁶ cells mL⁻¹ and were bubbled with the indicated concentrations of air for 24 h prior to preparation and analysis of total RNA. CO₂ was kept constant at 2%. Endogenous Cpx1 was probed as a positive control for the efficacy of the oxygen deprivation, and RbcS was probed as a control for loading (not shown). Copper-responsive expression of the same constructs was analyzed by Quinn et al. (2000a). Multiple, independent transformants were generated for each construct. The data shown are from a single representative transformant.

Figure 7

The crr1 mutant is blocked in the oxygen deficiency response. Total RNA isolated from the crr1 mutant and a wild-type strain (CRR1) grown under the indicated copper- (A) or oxygen-deficient (B) conditions was analyzed for Crd1, Cpx1, and Cyc6 expression. Total RNA from copper-deficient crr1 was analyzed for Ars2 expression in response to sulfur deficiency (C) or Ftr1 (encoding a ferric transporter) in response to iron deficiency (D). Comparable expression of these genes in wild-type cells is not shown. Total RNA was analyzed for Hyd1 expression (E) in wild-type versus crr1 cells. A time course of response to oxygen deprivation (achieved by transfer to 98% N₂/2% CO₂) is shown with Cyc6 expression as an internal reference.

Figure 8

Mercuric ions specifically block the oxygen deficiency response of Cpx1 and Cyc6. A, Wild-type cells were grown to 2 × 10⁶ cells mL⁻¹ and were divided into three subcultures. Each subculture was bubbled with 1% air (2% CO₂, balance N₂) for the indicated times and HgCl₂ was added to 10 μm final concentration at the indicated times. A, No added HgCl₂; B, HgCl₂ added at t = 0 h; C, HgCl₂ added 2 h after initiation of hypoxic treatment. B, Response of Tub2 (encoding β-tubulin). A culture of CC125 was grown to 2 × 10⁶ cells mL⁻¹ and was divided into six subcultures. Each subculture was bubbled with 1% air for the indicated times prior to sampling for RNA preparation. HgCl₂ was added to the final indicated concentrations after 2 h of treatment with 1% air. C, Response of Hyd1 (encoding Fe-hydrogenase). Mercuric chloride was added to a final concentration of 1 μm 2 h before or immediately prior to transfer to 0% air (2% CO₂/98% N₂; A and B). As a positive control, no addition was made before or after transfer to 0% air (2% CO₂/98% N₂; C).