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.

Copper delivery to the thylakoid lumen protein plastocyanin and the stromal enzyme Cu/Zn superoxide dismutase in chloroplasts is required for photosynthesis and oxidative stress protection. The copper delivery system in chloroplasts was characterized by analyzing the function of copper transporter genes in Arabidopsis thaliana. Two mutant alleles were identified of a previously uncharacterized gene, PAA2 (for P-type ATPase of Arabidopsis), which is required for efficient photosynthetic electron transport. PAA2 encodes a copper-transporting P-type ATPase with sequence similarity to PAA1, which functions in copper transport in chloroplasts. Both proteins localized to the chloroplast, as indicated by fusions to green fluorescent protein. The PAA1 fusions were found in the chloroplast periphery, whereas PAA2 fusions were localized in thylakoid membranes. The phenotypes of paa1 and paa2 mutants indicated that the two transporters have distinct functions: whereas both transporters are required for copper delivery to plastocyanin, copper delivery to the stroma is inhibited only in paa1 but not in paa2. The effects of paa1 and paa2 on superoxide dismutase isoform expression levels suggest that stromal copper levels regulate expression of the nuclear genes IRON SUPEROXIDE DISMUTASE1 and COPPER/ZINC SUPEROXIDE DISMUTASE2. A paa1 paa2 double mutant was seedling-lethal, underscoring the importance of copper to photosynthesis. We propose that PAA1 and PAA2 function sequentially in copper transport over the envelope and thylakoid membrane, respectively.