Manganese co-localizes with calcium and phosphorus in Chlamydomonas acidocalcisomes and is mobilized in manganese-deficient conditions.

Exposing cells to excess metal concentrations well beyond the cellular quota is a powerful tool for understanding the molecular mechanisms of metal homeostasis. Such improved understanding may enable bioengineering of organisms with improved nutrition and bioremediation capacity. We report here that Chlamydomonas reinhardtii can accumulate manganese (Mn) in proportion to extracellular supply, up to 30-fold greater than its typical quota and with remarkable tolerance. As visualized by X-ray fluorescence microscopy and nanoscale secondary ion MS (nanoSIMS), Mn largely co-localizes with phosphorus (P) and calcium (Ca), consistent with the Mn-accumulating site being an acidic vacuole, known as the acidocalcisome. Vacuolar Mn stores are accessible reserves that can be mobilized in Mn-deficient conditions to support algal growth. We noted that Mn accumulation depends on cellular polyphosphate (polyP) content, indicated by 1) a consistent failure of C. reinhardtii vtc1 mutant strains, which are deficient in polyphosphate synthesis, to accumulate Mn and 2) a drastic reduction of the Mn storage capacity in P-deficient cells. Rather surprisingly, X-ray absorption spectroscopy, EPR, and electron nuclear double resonance revealed that only little Mn2+ is stably complexed with polyP, indicating that polyP is not the final Mn ligand. We propose that polyPs are a critical component of Mn accumulation in Chlamydomonas by driving Mn relocation from the cytosol to acidocalcisomes. Within these structures, polyP may, in turn, escort vacuolar Mn to a number of storage ligands, including phosphate and phytate, and other, yet unidentified, compounds.

The proteome of copper, iron, zinc, and manganese micronutrient deficiency in Chlamydomonas reinhardtii.

Trace metals such as copper, iron, zinc, and manganese play important roles in several biochemical processes, including respiration and photosynthesis. Using a label-free, quantitative proteomics strategy (MS(E)), we examined the effect of deficiencies in these micronutrients on the soluble proteome of Chlamydomonas reinhardtii. We quantified >10(3) proteins with abundances within a dynamic range of 3 to 4 orders of magnitude and demonstrated statistically significant changes in ~200 proteins in each metal-deficient growth condition relative to nutrient-replete media. Through analysis of Pearson's coefficient, we also examined the correlation between protein abundance and transcript abundance (as determined via RNA-Seq analysis) and found moderate correlations under all nutritional states. Interestingly, in a subset of transcripts known to significantly change in abundance in metal-replete and metal-deficient conditions, the correlation to protein abundance is much stronger. Examples of new discoveries highlighted in this work include the accumulation of O(2) labile, anaerobiosis-related enzymes (Hyd1, Pfr1, and Hcp2) in copper-deficient cells; co-variation of Cgl78/Ycf54 and coprogen oxidase; the loss of various stromal and lumenal photosynthesis-related proteins, including plastocyanin, in iron-limited cells; a large accumulation (from undetectable amounts to over 1,000 zmol/cell) of two COG0523 domain-containing proteins in zinc-deficient cells; and the preservation of photosynthesis proteins in manganese-deficient cells despite known losses in photosynthetic function in this condition.

Systems biology approach in Chlamydomonas reveals connections between copper nutrition and multiple metabolic steps.

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.

A revised mineral nutrient supplement increases biomass and growth rate in Chlamydomonas reinhardtii.

Interest in exploiting algae as a biofuel source and the role of inorganic nutrient deficiency in inducing triacylglyceride (TAG) accumulation in cells necessitates a strategy to efficiently formulate species-specific culture media that can easily be manipulated. Using the reference organism Chlamydomonas reinhardtii, we tested the hypothesis that modeling trace element supplements after the cellular ionome would result in optimized cell growth. We determined the trace metal content of several commonly used Chlamydomonas strains in various culture conditions and developed a revised trace element solution to parallel these measurements. Comparison of cells growing in the revised supplement versus a traditional trace element solution revealed faster growth rates and higher maximum cell densities with the revised recipe. RNA-seq analysis of cultures growing in the traditional versus revised medium suggest that the variation in transcriptomes was smaller than that found between different wild-type strains grown in traditional Hutner's supplement. Visual observation did not reveal defects in cell motility or mating efficiency in the new supplement. Ni²âº-inducible expression from the CYC6 promoter remained a useful tool, albeit with an increased requirement for Ni²âº because of the introduction of an EDTA buffer system in the revised medium. Other advantages include more facile preparation of trace element stock solutions, a reduction in total chemical use, a more consistent batch-to-batch formulation and long-term stability (tested up to 5 years). Under the new growth regime, we analyzed cells growing under different macro- and micronutrient deficiencies. TAG accumulation in N deficiency is comparable in the new medium. Fe and Zn deficiency also induced TAG accumulation, as suggested by Nile Red staining. This approach can be used to efficiently optimize culture conditions for other algal species to improve growth and to assay cell physiology.

Comparison of the kinetics of iron release from a marine (Trichodesmium erythraeum) Dps protein and mammalian ferritin in the presence and absence of ligands.

The ferritin superfamily of iron storage proteins includes ferritin proper and Dps (DNA binding protein from starved cells) along with bacterioferritin. We examined the release of Fe from the Dps of Trichodesmium erythraeum (Dps(tery)) and compared it to the release of Fe from horse spleen ferritin (HoSF) under various conditions. Both desferrioxamine B (DFB), a Fe(III) chelator, and ascorbic acid were able to mobilize Fe from Dps(tery) at rates comparable to those observed for HoSF. The initial Fe release rate from both proteins increased linearly with the concentration of DFB, suggesting that the chelator binds to Fe in the protein. A small but significant rate obtained by extrapolation to zero concentration of DFB implies that Dps(tery) and HoSF might release Fe(III) spontaneously. A similar result was observed for HoSF in the presence of sulfoxine. In a different experiment, Fe(III) was transferred from holoferritin to apotransferrin across a dialysis membrane in the absence of chelator or reducing agent. The apparent spontaneous release of Fe from HoSF and Dps(tery) brings forth the hypothesis that the Fe core in Fe storage proteins might be continuously dissolving and re-precipitating in vivo, thus maintaining it in a highly reactive and bioavailable form.

Ni induces the CRR1-dependent regulon revealing overlap and distinction between hypoxia and Cu deficiency responses in Chlamydomonas reinhardtii.

The selectivity of metal sensors for a single metal ion is critical for cellular metal homeostasis. A suite of metal-responsive regulators is required to maintain a prescribed balance of metal ions ensuring that each apo-protein binds the correct metal. However, there are cases when non-essential metals ions disrupt proper metal sensing. An analysis of the Ni-responsive transcriptome of the green alga Chlamydomonas reinhardtii reveals that Ni artificially turns on the CRR1-dependent Cu-response regulon. Since this regulon also responds to hypoxia, a combinatorial transcriptome analysis was leveraged to gain insight into the mechanisms by which Ni interferes with the homeostatic regulation of Cu and oxygen status. Based on parallels with the effect of Ni on the hypoxic response in animals, we propose that a possible link between Cu, oxygen and Ni sensing is an as yet uncharacterized prolyl hydroxylase that regulates a co-activator of CRR1. This analysis also identified transcriptional responses to the pharmacological activation of the Cu-deficiency regulon. Although the Ni-responsive CRR1 regulon is composed of 56 genes (defined as the primary response), 259 transcripts responded to Ni treatment only when a copy of the wild-type CRR1 gene was present. The genome-wide impact of CRR1 target genes on the transcriptome was also evident from the 210 transcripts that were at least 2-fold higher in the crr1 strain, where the abundance of many CRR1 targets was suppressed. Additionally, we identified 120 transcripts that responded to Ni independent of CRR1 function. The putative functions of the proteins encoded by these transcripts suggest that high Ni results in protein damage.