Copper Delivery by the Copper Chaperone for Chloroplast and Cytosolic Copper/Zinc-Superoxide Dismutases: Regulation and Unexpected Phenotypes in an Arabidopsis Mutant

Abstract

Copper (Cu) is an important mineral nutrient found in chloroplasts as a cofactor associated with plastocyanin and Cu/Zn superoxide dismutase (Cu/ZnSOD). Superoxide dismutases are metallo-enzymes found in most oxygenic organisms with proposed roles in reducing oxidative stress. Several recent studies in Arabidopsis have shown that microRNAs and a SQUAMOSA promoter binding protein-like7 (SPL7) transcription factor function to down-regulate the expression of many Cu-proteins, including Cu/ZnSOD in both plastids and the cytosol, during growth on low Cu. Plants contain the Cu Chaperone for SOD (CCS) that delivers Cu to Cu/ZnSODs, and, in Arabidopsis, both cytosolic and plastidic CCS versions are encoded by one gene. In this study, we demonstrate that Arabidopsis CCS transcript levels are regulated by Cu, mediated by microRNA 398 that was not previously predicted to target CCS. The microRNA target site is conserved in CCS of Oryza sativa. The data suggest that Cu-regulated microRNAs may have more mRNA targets than was previously predicted. A CCS null mutant has no measurable SOD activity in the chloroplast and cytosol, indicating an absolute requirement for CCS. When the CCS null mutant was grown on high Cu media, it lacked both Fe superoxide dismutase (FeSOD) and Cu/ZnSOD activity. However, this did not lead to a visual phenotype and no photosynthetic deficiencies were detected, even after high light stress. These results indicate that Cu/ZnSOD is not a pivotal component of the photosynthetic anti-oxidant system during growth in laboratory conditions.

Copper Chaperone-Dependent and -Independent Activation of Three Copper-Zinc Superoxide Dismutase Homologs Localized in Different Cellular Compartments in Arabidopsis

“…Therefore, the lower germination rate of Atcsd1 was possibly due to the complete absence of CSD1 activity in the cytoplasm. In addition, current studies of SODs indicate that the level of SOD activity required for normal growth is much lower than the actual activity level measured (Cohu et al, 2009 (Cohu et al, 2009).…”

“…The CCS interacts with CSD, assisting in Cu incorporation and catalysis of disulfide bond formation, thereby resulting in an active CSD (Casareno et al, 1998;Lamb et al, 2001;Brown et al, 2004;Furukawa et al, 2004). In Arabidopsis, a single AtCCS gene product activated CSDs in the cytoplasm and chloroplast (Chu et al, 2005); however, the phenotype of the AtCCS-knockout mutant (Atccs) was found to be normal (Chu et al, 2005;Cohu et al, 2009). Similarly, a CCS-independent activation pathway for CSD was found in mice (Wong et al, 2000).…”

Functional Characterization of the GATA Transcription Factors GNC and CGA1 Reveals Their Key Role in Chloroplast Development, Growth, and Division in Arabidopsis

“…Prior to chlorophyll fluorescence imaging analysis, plants were dark adapted for 30 min. Analysis was performed as described (Cohu et al, 2009). The F v /F m and F PSII were calculated as described (Maxwell and Johnson, 2000).…”

Spatiotemporal Analysis of Copper Homeostasis in Populus trichocarpa Reveals an Integrated Molecular Remodeling for a Preferential Allocation of Copper to Plastocyanin in the Chloroplasts of Developing Leaves

“…This idea was based on the observed Cu miRNA-mediated down-regulation of several Cu proteins in Arabidopsis (Abdel-Ghany et al, 2005;Yamasaki et al, 2007;Abdel-Ghany and Pilon 2008;Dugas and Bartel, 2008;Cohu et al, 2009) as well as in Brassica species, tomato (Solanum lycopersicum), maize (Zea mays), and rice (Oryza sativa; Cohu and Pilon, 2007). However, in Arabidopsis, PC2 protein was reported to be strongly affected by Cu (Abdel-Ghany 2009), and PC protein levels were also affected by Cu in poplar (Fig.…”

“…SPL7 is required for the up-regulation of cellular Cu uptake and assimilation mechanisms, which is highlighted by the up-regulation of the expression of the plasma membrane COPT1 Cu transporter (Yamasaki et al, 2009). On low Cu, SPL7 also is required for expression of the so-called Cu microRNAs (miRNAs; Burkhead et al, 2009), which in turn down-regulate transcripts for the Cu proteins CCS, Cu/ZnSOD1 (CSD1), and CSD2 (targets of miR398), plantacyanin (target of miR408), and several laccases (targets of miR397, miR408, and miR857; Sunkar et al, 2006;Yamasaki et al, 2007;Abdel-Ghany and Pilon, 2008;Dugas and Bartel, 2008;Cohu et al, 2009;Yamasaki et al, 2009). A Cu economy model has been hypothesized in which the miRNA-mediated mechanism saves Cu for the most essential Cu proteins, such as COX and PC, during impending deficiency (Burkhead et al, 2009, and refs.…”