Contents Summary  799 Introduction  800 The origins of Cu homeostasis  800 Copper homeostasis in unicellular photosynthetic model organisms  801 Functions of Cu in plants  802 Typical levels of Cu in plants, deficiency and toxicity  802 Copper abundance in soils and appropriate Cu concentrations in media  804 Uptake in the root and distribution to aerial tissues  804 Uptake in the shoot symplast, redistribution of Cu during flowering, seed set and senescence  806 Cu delivery inside the cell  806 Regulation of Cu homeostasis  809 Conclusions and outlook  811 Acknowledgements  811 References  811 Summary Copper (Cu) is a cofactor in proteins that are involved in electron transfer reactions and is an essential micronutrient for plants. Copper delivery is accomplished by the concerted action of a set of evolutionarily conserved transporters and metallochaperones. As a result of regulation of transporters in the root and the rarity of natural soils with high Cu levels, very few plants in nature will experience Cu in toxic excess in their tissues. However, low Cu bioavailability can limit plant productivity and plants have an interesting response to impending Cu deficiency, which is regulated by an evolutionarily conserved master switch. When Cu supply is insufficient, systems to increase uptake are activated and the available Cu is utilized with economy. A number of Cu‐regulated small RNA molecules, the Cu‐microRNAs, are used to downregulate Cu proteins that are seemingly not essential. On low Cu, the Cu‐microRNAs are upregulated by the master Cu‐responsive transcription factor SPL7, which also activates expression of genes involved in Cu assimilation. This regulation allows the most important proteins, which are required for photo‐autotrophic growth, to remain active over a wide range of Cu concentrations and this should broaden the range where plants can thrive.

In plants, copper is an essential micronutrient required for photosynthesis. Two of the most abundant copper proteins, plastocyanin and copper/zinc superoxide dismutase, are found in chloroplasts. Whereas plastocyanin is essential for photo-autotrophic growth, copper/zinc superoxide dismutase is dispensable and in plastids can be replaced by an iron superoxide dismutase when copper is limiting. The down-regulation of copper/zinc superoxide dismutase expression in response to low copper involves a microRNA, miR398. Interestingly, in Arabidopsis and other plants, three additional microRNA families, miR397, miR408, and miR857, are predicted to target the transcripts for the copper protein plantacyanin and members of the laccase copper protein family. We confirmed the predicted targets of miR397, miR408, and miR857 experimentally by cleavage site analysis. To study the spatial expression pattern of these microRNAs and the effect of copper on their expression, we analyzed Arabidopsis grown hydroponically on different copper regimes. On low amounts of copper the plants accumulated miR397, miR408, and miR857. The microRNA expression pattern was negatively correlated with the accumulation of transcripts for plantacyanin and laccases. Furthermore, the expression of other laccases that are not predicted targets for known microRNAs was similarly regulated in response to copper. For some of these laccases, the regulation was disrupted in a microRNA maturation mutant (hen1-1), suggesting the presence of other copper-regulated microRNAs. Thus, in Arabidopsis, microRNA-mediated down-regulation is a general mechanism to regulate nonessential copper proteins. We propose that this mechanism allows plants to save copper for the most essential functions during limited copper supply.

Major copper proteins in the cytoplasm of plant cells are plastocyanin, copper/zinc superoxide dismutase, and cytochrome c oxidase. Under copper limited conditions, expression of copper/ zinc superoxide dismutase is down-regulated and the protein is replaced by iron superoxide dismutase in chloroplasts. We present evidence that a micro-RNA, miR398, mediates this regulation in Arabidopsis thaliana, by directing the degradation of copper/zinc superoxide dismutase mRNA when copper is limited. Sequence analysis indicated that the transcripts encoding cytosolic copper/zinc superoxide dismutase and COX5b-1, a subunit of the mitochondrial cytochrome c oxidase, are also targeted by miR398. This regulation via miR398 takes place in response to changes in a low range of copper levels (0.2-0.5 M), indicating that miR398 is involved in a response to copper limitation. On the other hand, another major copper protein, plastocyanin, which is involved in photosynthetic electron flow and is essential in higher plants, was not regulated via miR398. We propose that miR398 is a key factor in copper homeostasis in plants and regulates the stability of mRNAs of major copper proteins under copper-limited conditions.

Degradation of misfolded secretory proteins has long been assumed to occur in the lumen of the endoplasmic reticulum (ER). Recent evidence, however, suggests that such proteins are instead degraded by proteasomes in the cytosol, although it remains unclear how the proteins are transported out of the ER. Here we provide the first genetic evidence that Sec61p, the pore-forming subunit of the protein translocation channel in the ER membrane, is directly involved in the export of misfolded secretory proteins. We describe two novel mutants in yeast Sec61p that are cold-sensitive for import into the ER in both intact yeast cells and a cell-free system. Microsomes derived from these mutants are defective in exporting misfolded secretory proteins. These proteins become trapped in the ER and are associated with Sec61p. We conclude that misfolded secretory proteins are exported for degradation from the ER to the cytosol via channels formed by Sec61p.

Copper (Cu) is an essential trace element with important roles as a cofactor in many plant functions, including photosynthesis. However, free Cu ions can cause toxicity, necessitating precise Cu delivery systems. Relatively little is known about Cu transport in plant cells, and no components of the Cu transport machinery in chloroplasts have been identified previously. Cu transport into chloroplasts provides the cofactor for the stromal enzyme copper/zinc superoxide dismutase (Cu/ZnSOD) and for the thylakoid lumen protein plastocyanin, which functions in photosynthetic electron transport from the cytochrome b 6 f complex to photosystem I. Here, we characterized six Arabidopsis mutants that are defective in the PAA1 gene, which encodes a member of the metal-transporting P-type ATPase family with a functional N-terminal chloroplast transit peptide. paa1 mutants exhibited a high-chlorophyll-fluorescence phenotype as a result of an impairment of photosynthetic electron transport that could be ascribed to decreased levels of holoplastocyanin. The paa1-1 mutant had a lower chloroplast Cu content, despite having wild-type levels in leaves. The electron transport defect of paa1 mutants was evident on medium containing Ͻ 1 M Cu, but it was suppressed by the addition of 10 M Cu. Chloroplastic Cu/ZnSOD activity also was reduced in paa1 mutants, suggesting that PAA1 mediates Cu transfer across the plastid envelope. Thus, PAA1 is a critical component of a Cu transport system in chloroplasts responsible for cofactor delivery to plastocyanin and Cu/ZnSOD.