From economy to luxury: Copper homeostasis in Chlamydomonas and other algae

Abstract

Plastocyanin and cytochrome c₆, abundant proteins in photosynthesis, are readouts for cellular copper status in Chlamydomonas and other algae. Their accumulation is controlled by a transcription factor copper response regulator (CRR1). The replacement of copper-containing plastocyanin with heme-containing cytochrome c₆ spares copper and permits preferential copper (re)-allocation to cytochrome oxidase. Under copper-replete situations, the quota depends on abundance of various cuproproteins and is tightly regulated, except under zinc-deficiency where acidocalcisomes over-accumulate Cu(I). CRR1 has a transcriptional activation domain, a Zn-dependent DNA binding SBP-domain with a nuclear localization signal, and a C-terminal Cys-rich region that represses the zinc regulon. CRR1 activates >60 genes in Chlamydomonas through GTAC-containing CuREs; transcriptome differences are recapitulated in the proteome. The differentially-expressed genes encode assimilatory copper transporters of the CTR/SLC31 family including a novel soluble molecule, redox enzymes in the tetrapyrrole pathway that promote chlorophyll biosynthesis and photosystem 1 accumulation, and other oxygen-dependent enzymes, which may influence thylakoid membrane lipids, specifically polyunsaturated galactolipids and γ-tocopherol. CRR1 also down-regulates 2 proteins in Chlamydomonas: for plastocyanin, by activation of proteolysis, while for the di‑iron subunit of the cyclase in chlorophyll biosynthesis, through activation of an upstream promoter that generates a poorly-translated 5' extended transcript containing multiple short ORFs that inhibit translation. The functions of many CRR1-target genes are unknown, and the copper protein inventory in Chlamydomonas includes several whose functions are unexplored. The comprehensive picture of cuproproteins and copper homeostasis in this system is well-suited for reverse genetic analyses of these under-investigated components in copper biology.

Keywords: Chlamydomonas; Chlorophyte algae; Chloroplast; Copper nutrition; Ferredoxin; Photosynthesis.

Figure 1:. The elemental composition of Chlamydomonas.

Laboratory-grown wild-type cells were grown in nutrient-replete conditions. C and N were determined on a Shimadzu TOC/TN analyzer (dashed outline), and other elements by ICP-MS/MS on an Agilent 8800 (solid outline). A total of 12 samples was analyzed from wild-type Chlamydomonas laboratory strain CC-4532. The distribution is summarized in boxplots, where each horizontal line represents the 10, 25, 50, 75 and 90% quantile (from bottom to top).

Figure 2:. Increased bio-availability of copper in an oxygenated biosphere.

Elemental estimates are based on the chemical characteristics of ancient sediments [98].

Figure 3:. Copper conditional phenotype of a plastocyanin-deficient mutant.

Algal cells were grown photoautotrophically on plates supplemented with copper (+Cu) or not (−Cu). wt = wild-type for plastocyanin synthesis, pcy1 = no synthesis of plastocyanin because of a frame-shift mutation [9].

Figure 4:. CRR1 schematic and family analysis.

A, Putative functional domains discussed in the text are indicated. Med15 = blue, SBP = dark pink, extended SBP = light pink, ankyrin repeats = purple, C-terminal Cys-rich region = turquoise, green= nuclear localization signal. The numbers on the top indicate amino acid positions in CRR1. The position of mutations in crr1 mutants are indicated. Sequence logos for conserved motifs identified within the extended SBP region and their locations are shown. Homologs containing these motifs are highlighted in panel C with a triangle. The Cys-rich region resembles metallothionein and is found also in CRR1 from Chlamydomonas eustigma, Volvox carteri, Gonium pectorale, Tetrabaena socialis, Raphidocelis subcapitata, Tetradesmus obliquus and Chromochloris zofingiensis. Two conserved motifs identified from this region are shown as sequence logos. B, Phylogenetic tree of the SPB domain from green algae and land plants. Protein sequences were aligned using NCBI’s Cobalt aligner [99]. An edited alignment encompassing just the SBP domain was used to build a phylogenetic tree with FastTree [100], using 1000 bootstrap replicates, on the CIPRES Science Gateway [101]. The tree was visualized with iTol [102] and branches with a bootstrap value less than 0.5 were deleted. Branch lengths are ignored. Leaves are colored by taxonomy according to legend. Based on this analysis, the family can be divided into two groups. Group B only contains algal sequences, while group A contains algal homologs (with the exception of prasinophytes) and land plant homologs. Chlamydomonas homologs in group B and CRR1 are labeled. Chlamydomonas homologs from group A can be found in panel C. C, Unrooted phylogenetic tree of sequences from group A, which contains CRR1 from Chlamydomonas and SPL7 from Arabidopsis, as well as the previously-characterized nitrogen response regulator, NRR1 [103], from Chlamydomonas. Full-length sequences from group A were realigned using MAFFT [104] then the full-length alignment was used to build a phylogenetic tree as in panel B. Each circle represents an organism as indicated in the legend and the background is colored by taxonomy.

Figure 5 –. Altered distribution of copper depending on copper nutrition status.

The copper content of Chlamydomonas cells in replete vs. deficient medium was measured by ICP-MS. The abundances of copper proteins (in units of millions of molecules per cell) were estimated (left hand side of table) by quantitative proteomics using the proteomic ruler method [105]. The contribution of each protein to the copper quota was derived based on the number of copper binding sites (middle column) in each protein to yield the amount of copper (in millions of atoms) associated with that protein in each cell (right hand side of table). The distribution of total cellular copper (from ICP-MS) amongst the 3 most abundant cuproproteins (light grey = plastocyanin (PC), grey = Cyt oxidase (COX) and dark grey = ferroxidase (FOX)) in Chlamydomonas is presented within the bar and also as a pie chart where the area reflects the amount of copper associated with each protein, and white represents the balance. Note that the cells were grown in iron-replete conditions, where the ferroxidase abundance is low.

Figure 6.. Co-occurrence of SBP-domains, CRR1 and Cu-deficiency targets in plant and green algal proteomes.

Proteins were predicted from public whole genome sequencing projects (as of June 2020). Assignments were based on reciprocal best hits with the Chlamydomonas reinhardtii homologs. In many cases faulty or absent gene models were corrected by searching the genome with tblastn and using comparisons with homologs from closely related species. Open circles signify the absence of a gene/protein. Absence of a protein may mean that it was never there or never acquired, or that it was lost, or that the genome assembly is incomplete. Copy # of paralogs within a species is indicated within the filled circles using a grey scale. The “CRR1” designation in the figure was based on prediction of CRR1 orthologs using the tree in Figure 4C. However, we note that not all orthologs share the same motifs with CRR1, such as the extended SBP domain or the ankyrin motifs. These absence could be due to incorrect gene models or functional divergence. SBP Group A and Group B proteins may be functional homologs of CRR1 in other algae, as is the case for SPL7 in Arabidopsis. Cu-deficiency targets include PCY1, encoding a blue copper protein that transfers electrons from cytochrome b₆f to oxidized PSI. Heme-containing cyt c₆ (encoded by CYC6) functionally substitutes for PCY1 in Cu-deficient cells. In Chlamydomonas, the genes for Mg-protoporphyrin IX monomethylester cyclase components CHL27 (CTH1 & CRD1 parologs) and CGL78 are all regulated by CRR1 in response to Cu-availability. FDX5 and FDX6, both of whose genes are regulated by CRR1, may provide electrons for the cyclase reaction. Potential pseudogenes resulting from gene fusions, deletions, inversions and frame-shifts are included in the CHL27 copy # for Monoraphidium neglectum, Raphidocelis subcapitata, Chlorella variabilis and Micractinium conductrix. Abbreviations and gene product names: A, SBP Group A proteins; B, SBP Group B proteins; CRR1, copper response regulator; PCY1, plastocyanin; CYC6, cytochrome c₆; CHL27, Mg-protoporphyrin IX monomethylester cyclase; CGL78, conserved in green lineage 78 (component of the Mg-protoporphyrin IX monomethylester cyclase); FDX5, ferredoxin 5; FDX6, ferredoxin 6.