How do plants respond to copper deficiency?

Abstract

The transition metal copper is essential for all organisms yet excess copper is toxic because of production of free radicals via its free form. Therefore, the levels of copper are precisely regulated in a cell. Under copper depleted conditions, the expression of Cu/Zn superoxide dismutase (SOD) is downregulated and its function is compensated by Fe SOD in chloroplasts of higher plants. We presented evidence that a microRNA, miR398, is involved in this downregulation of Cu/Zn SOD genes in Arabidopsis thaliana when grown at low copper levels, corresponding to less than 1 µM Cu in tissue culture media. However, a green alga, Chlamydomonas reinhardtii, adjusts to copper depletion by modifying the photosynthetic apparatus from copper containing plastocyanin to iron containing cytochrome c₆. During evolution plants modified one of the main strategies to respond to copper deficiency probably to adapt to different metal environments.

Copper is an essential micronutrient and is involved in several metabolic processes.¹ In higher plants, the most abundant copper protein is plastocyanin, which is involved in the photosynthetic electron transport in the thylakoid lumen of chloroplasts. Another major copper protein, Cu/Zn SOD, localizes to the cytoplasm (CSD1), stroma of chloroplasts (CSD2) and also peroxisomes (CSD3), and is involved in the scavenging of reactive oxygen species.² Despite its physiological importance, excess copper is toxic for plants because of its potential participation in the Fenton reaction. To minimize the damage by excess copper and also respond to copper deficiency, higher plants have several strategies, including the regulation of copper uptake in root cells,³ strict copper trafficking via P-type ATPases and copper chaperones^4–9 or regulation of the levels of copper proteins in response to a change in the metal availability.^10,11 In addition plants respond to copper deficiency by expressing the alternative iron proteins which complement the function of copper proteins.¹²

Among photosynthetic eukaryotes, the unicellular green alga, Chlamydomonas reinhardtii, is best studied with respect to the molecular mechanism of copper homeostasis.¹³ Under low copper conditions, Chlamydomonas degrades plastocyanin and its function is replaced by cytchrome c₆ containing iron.¹² A transcriptional factor, Crr1, plays a pivotal role in switching the photosynthetic machinery and probably also in copper sensing.¹⁴ However, higher plants are unlikely to encode the functional homolog of the algal cytochrome c₆.¹⁵ How do higher plants respond to copper deficiency?

In Arabidopsis, the expression of CSD1 and CSD2 is downregulated in copper deficient conditions.⁵ MicroRNA, miR398, is involved in this downregulation of both Cu/ZnSOD genes.¹⁰ While microRNAs extensively regulate a variety of developmental processes,¹⁶ they are also involved in responses to the environmental stresses including deficiency in sulfur¹⁷ and phosphate.¹⁸ miR398 is expressed only in low copper conditions¹⁰ and directly involved in the degradation of CSD1 and CSD2 mRNA.^10,19 Consequently, this regulation allows limited copper to be preferentially transferred to plastocyanin whose expression is independent of the regulation via miR398. Even in the absence of the functional homolog of algal cytochrome c₆, higher plants can sustain photosynthesis in the low copper conditions. Meanwhile, the function of CSD2 is replaced by Fe SOD (FSD1). This switching between Cu/Zn SOD and Fe SOD takes place at the range of 0.1–1 µM CuSO₄ in agar-solidified medium.¹⁰ Arabidopsis seedlings grown in the soil used in our laboratory express both Cu/Zn and Fe SODs (data not shown), suggesting that the soil contains copper ion equivalent to the range of 0.1–1 µM in the medium. Arabidopsis seedlings can perceive a change in copper concentration in the physiological range for maintaining copper homeostasis. The rice and poplar genomes encode microRNA which is homologous to miR398, suggesting a conserved mechanism by which higher plants adapt to copper deficiency.

During evolution from algae to higher plants, one of major strategies to adapt to copper deficiency was altered from the system switching plastocycnin to cytochrome c₆ to that switching Cu/Zn SOD to Fe SOD. We are interested in the strategy in a moss, Physcomitrella patens, which is a model plant of the primitive land plants. Based on the genome information (genome.jgi-psf.org//Phypa1_1/Phypa1_1.home.html), the P. patens genome encodes at least three Cu/Zn SODs, two chloroplast forms and one cytosolic form. Expression of these CSD genes was analyzed in P. patens cultured on the agar medium containing various concentrations of CuSO₄ (Yamasaki et al., unpublished data). Both RT-PCR and protein blot analysis indicate that the gene expression is differently regulated across the boundary existing between 0.05 µM and 0.1 µM CuSO₄. In contrast, the plastocyanin gene is constitutively expressed at any copper concentrations, as in Arabidopsis. However, a genome wide analysis of microRNA has not identified the microRNA similar to Arabidopsis miR398 in the P. patens genome. It is possible that the expression of P. patens CSDs is regulated at transcriptional level. The P. patens genome is unlikely to encode a functional homolog of algal cytochrome c₆, and plastocyanin may be essential for photosynthesis even under copper deficient conditions. Taken together all the information, P. patens is likely to respond to copper deficiency with the similar strategy of higher plants, rather than that of algae.

Information from three model plants may not be sufficient to conclude something definitively, but we hypothesize that plants acquired the higher plant-type strategy to respond to copper deficiency in the early evolution of land plants (Fig. 1). This is probably related to the evolutionary event that plastocyanin rather than cytochrome c₆ was fixed as an electron carrier for photosynthetic electron transport of land plants. Plastocyanin is also essential for the oceanic diatom Thalassiosira oceanica, although other coastal species lack plastocyanin and rely on the function of cytochrome c₆.²⁰ In the open sea, iron levels are very low and copper is relatively more available. The low availability of iron may have led to a selection for the use of plastocyanin in both the land plants and in T. oceanica. In the land plants, which can also express a Cu/Zn SOD this could have lead to the evolution of the regulation of SOD genes. Unlike Chlamydomonas, the land plants do not use a copper-dependent iron uptake system,²¹ which is well conserved including animals, and this may allow plants to save copper for photosynthesis. How did the higher plants incorporate the function of microRNA into the regulatory process and how is the regulation via the microRNA related to the other strategies for metal homeostasis? Characterization of three model plants, Chlamydomonas, P. patens and Arabidopsis, may lead to answers to these questions.

Figure 1.

Adaptations to respond to variable copper supply in plants. When copper is limiting, Chlamydmonas degrades plastocyanin (PC) and its function is replaced by cytochrome c₆ (CYC6). In contrast to this algal strategy, Arabidopsis switches from the use of Cu/Zn SoD to iron SoD in response to copper deficiency and this switch involves the function of miR398. The strategy of Physcomitrella patens is likely to be similar to that of Arabidopsis, although the involvement microRNA is presently unclear.

Addendum to: Yamasaki H, Abdel-Ghany SE, Cohu CM, Kobayashi Y, Shikanai T, Pilon M. Regulation of copper homeostasis by micro-RNA in Arabidopsis. J Biol Chem. 2007;282:16369–16378. doi: 10.1074/jbc.M700138200.