title: Systems biology approach in Chlamydomonas reveals connections between copper nutrition and multiple metabolic steps.
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.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095, USA.
authors.forename: Madeli
authors.initials: M
authors.lastname: Castruita
authors.forename: David
authors.initials: D
authors.lastname: Casero
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Karpowicz
authors.forename: Janette
authors.initials: J
authors.lastname: Kropat
authors.forename: Astrid
authors.initials: A
authors.lastname: Vieler
authors.forename: Scott I
authors.initials: SI
authors.lastname: Hsieh
authors.forename: Weihong
authors.initials: W
authors.lastname: Yan
authors.forename: Shawn
authors.initials: S
authors.lastname: Cokus
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Loo
authors.forename: Christoph
authors.initials: C
authors.lastname: Benning
authors.forename: Matteo
authors.initials: M
authors.lastname: Pellegrini
authors.forename: Sabeeha S
authors.initials: SS
authors.lastname: Merchant
mesh_terms: D052818:Autotrophic Processes / Q000235:genetics; D001483:Base Sequence; D002696:Chlamydomonas / Q000235:genetics* / Q000378:metabolism*; D003300:Copper / Q000172:deficiency / Q000378:metabolism*; D020869:Gene Expression Profiling; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant / Q000235:genetics; D056426:Genetic Loci / Q000235:genetics; D052836:Heterotrophic Processes / Q000235:genetics; D008660:Metabolism / Q000235:genetics*; D008969:Molecular Sequence Data; D009747:Nutritional Physiological Phenomena / Q000235:genetics*; D010084:Oxidation-Reduction; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D017434:Protein Structure, Tertiary; D020543:Proteome / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D049490:Systems Biology
chemical_list: D010940:Plant Proteins; D020543:Proteome; D012333:RNA, Messenger; D018749:RNA, Plant; D003300:Copper