Abstract
In this work, we query the Chlamydomonas reinhardtii copper regulon at a whole-genome level. Our RNA-Seq data simulation and analysis pipeline validated a 2-fold cutoff and 10 RPKM (reads per kilobase of mappable length per million mapped reads) (~1 mRNA per cell) to reveal 63 CRR1 targets plus another 86 copper-responsive genes. Proteomic and immunoblot analyses captured 25% of the corresponding proteins, whose abundance was also dependent on copper nutrition, validating transcriptional regulation as a major control mechanism for copper signaling in Chlamydomonas. The impact of copper deficiency on the expression of several O₂-dependent enzymes included steps in lipid modification pathways. Quantitative lipid profiles indicated increased polyunsaturation of fatty acids on thylakoid membrane digalactosyldiglycerides, indicating a global impact of copper deficiency on the photosynthetic apparatus. Discovery of a putative plastid copper chaperone and a membrane protease in the thylakoid suggest a mechanism for blocking copper utilization in the chloroplast. We also found an example of copper sparing in the N assimilation pathway: the replacement of copper amine oxidase by a flavin-dependent backup enzyme. Forty percent of the targets are previously uncharacterized proteins, indicating considerable potential for new discovery in the biology of copper.
Highlights
Copper is an essential nutrient for all forms of aerobic life because of its role as a redox cofactor, especially in reactions involving oxygen chemistry, such as electron transfer during respiration and photosynthesis, neutralization of superoxide, and oxygenation of organic molecules
Since each cDNA is sequenced only once, the 17-nucleotide sequence tags are recovered in proportion to the abundance of the cDNA, which in turn represents the abundance of the mRNA
The sequence tags are mapped to the genome and assigned to annotated genes, and the number of reads correlates with transcript abundance
Summary
Copper is an essential nutrient for all forms of aerobic life because of its role as a redox cofactor, especially in reactions involving oxygen chemistry, such as electron transfer during respiration and photosynthesis, neutralization of superoxide, and oxygenation of organic molecules. Chlamydomonas reinhardtii is an excellent reference organism for understanding metabolic acclimation to copper deficiency because, in addition to its well-characterized genetics and genomics, it grows in a simple salts medium from which it is easy to reduce the copper content (Quinn and Merchant, 1998; Merchant et al, 2006). Plastocyanin is located in the chloroplast and functions as an electron carrier in photosynthesis, a ferroxidase on the plasma membrane functions in high affinity iron uptake, and cytochrome oxidase in the mitochondrion functions in respiration (Hanikenne et al, 2009)
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