Systems analysis of Chlamydomonas Cu nutrition reveals connections between Cu and multiple O2-dependent metabolic steps

We used digital gene expression (NlaIII sequence tags) and RNA-Seq to compare the transcriptomes of Cu-replete vs. Cu–deficient Chlamydomonas wild-type cells to reveal dozens of mRNAs whose abundance is modified. Half of the corresponding genes are targets of CRR1, a master regulator of nutritional copper sensing, and are associated with candidate CRR1 binding sites. The targets include many plastid-localized proteins, like FDX5 encoding a ferredoxin isoform, and CGL78, encoding a protein conserved in the green lineage, indicative of modified plastid metabolism. Immunoblot analysis and proteome profiles recapitulate the transcriptome profiles. New evidence for Cu sparing is suggested by up-regulation of AOF1 encoding a copper-independent but flavin-dependent amine oxidase and down-regulation of two metal- binding proteins. Genes encoding redox proteins, many of which function in lipid metabolism, are over-represented, which is compatible with the role of Cu in biology. Lipid profiles indicate a CRR1-dependent increase in Cu-deficient cells in the proportion of unsaturated (16:2, 16:3, 16:4, 18:2) fatty acids at the expense of the more saturated (16:0, 16:1, 18:0) precursors, especially on plastid galactolipids, which validates the increased expression of acyl-ACP and plastid-localized w-6 desaturases. CRR1-independent changes in the transcriptome suggest a role for Cu in oxygen sensing in Chlamydomonas. Sampling of Chlamydomonas CC-1021 (2137) and crr1-2, crr1:CRR1 mutant cells (the mutant is knock-down for the transcription factor crr1, which plays a key role in the transcriptional response to copper levels) cultivated in TAP or minimal medium under Cu-sufficient (control) and Cu-defficient conditions. poly-A purification, NlaIII digestion/random fragmentation