Even subtle modifications in growth conditions elicit acclimation responses affecting the molecular and elemental makeup of organisms, both in the laboratory and in natural habitats. We systematically explored the effect of temperature, pH, nutrient availability, culture density, and access to CO₂ and O₂ in laboratory-grown algal cultures on growth rate, the ionome, and the ability to accumulate Fe. We found algal cells accumulate Fe in alkaline conditions, even more so when excess Fe is present, coinciding with a reduced growth rate. Using a combination of Fe-specific dyes, X-ray fluorescence microscopy, and NanoSIMS, we show that the alkaline-accumulated Fe was intracellularly sequestered into acidocalcisomes, which are localized towards the periphery of the cells. At high photon flux densities, Zn and Ca specifically over-accumulate, while Zn alone accumulates at low temperatures. The impact of aeration was probed by reducing shaking speeds and changing vessel fill levels; the former increased the Cu quota of cultures, the latter resulted in a reduction in P, Ca, and Mn at low fill levels. Trace element quotas were also affected in the stationary phase, where specifically Fe, Cu, and Zn accumulate. Cu accumulation here depends inversely on the Fe concentration of the medium. Individual laboratory strains accumulate Ca, P, and Cu to different levels. All together, we identified a set of specific changes to growth rate, elemental composition, and the capacity to store Fe in response to subtle differences in culturing conditions of Chlamydomonas, affecting experimental reproducibility. Accordingly, we recommend that these variables be recorded and reported as associated metadata.

Growth of Chlamydomonas reinhardtii in zinc (Zn) limited medium leads to disruption of copper (Cu) homeostasis, resulting in up to 40-fold Cu over-accumulation relative to its typical Cu quota. We show that Chlamydomonas controls its Cu quota by balancing Cu import and export, which is disrupted in a Zn deficient cell, thus establishing a mechanistic connection between Cu and Zn homeostasis. Transcriptomics, proteomics and elemental profiling revealed that Zn-limited Chlamydomonas cells up-regulate a subset of genes encoding "first responder" proteins involved in sulfur (S) assimilation and consequently accumulate more intracellular S, which is incorporated into L-cysteine, γ-glutamylcysteine, and homocysteine. Most prominently, in the absence of Zn, free L-cysteine is increased ∼80-fold, corresponding to ∼2.8 × 109 molecules/cell. Interestingly, classic S-containing metal binding ligands like glutathione and phytochelatins do not increase. X-ray fluorescence microscopy showed foci of S accumulation in Zn-limited cells that co-localize with Cu, phosphorus and calcium, consistent with Cu-thiol complexes in the acidocalcisome, the site of Cu(I) accumulation. Notably, cells that have been previously starved for Cu do not accumulate S or Cys, causally connecting cysteine synthesis with Cu accumulation. We suggest that cysteine is an in vivo Cu(I) ligand, perhaps ancestral, that buffers cytosolic Cu.

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.