In Vitro Evolution and Preliminary Characterization of a Cadmium-Resistant Population of Chlamydomonas reinhardtii

Overview

Nagel and Voigt (Universität Hamburg; Institut für Biochemie und Lebensmittelchemie and Institut für Allgemeine Botanik und Botanischer Garten) describe the laboratory selection of a cadmium-tolerant population (CW15-Cdr) from the cadmium-sensitive cell-wall-deficient Chlamydomonas reinhardtii mutant CW-15, and compare cadmium uptake and intracellular cadmium distribution between the parent and the tolerant derivative to ask whether tolerance is mediated by the phytochelatin/cadmium-binding-peptide (CBP) detoxification system. The paper is a short three-page Notes article that reports a single 48-hour radiolabel experiment (29 μM 109CdCl2, 0.25 MBq of 109CdCl2) and ancillary growth, chlorophyll, and starch measurements. The headline finding is that the tolerant strain takes up more total cadmium (Cd) than the sensitive strain (220 ± 25 vs 190 ± 25 kcpm per 10^9 cells) but binds less of it to the CBP fraction (25,000 ± 5,000 cpm/10^9 cells, ~39% of cytosolic Cd, vs 45,000 ± 3,750 cpm/10^9 cells, ~67%), supporting the authors’ conclusion that Cd tolerance in CW15-Cdr is driven by genetically determined alterations of metabolism (specifically the photosystem II/chloroplast pathway) rather than by an enhanced phytochelatin-mediated detoxification system. In the wiki, this source is mechanistic background for Cadmium on the limits of the phytochelatin paradigm in unicellular algae; it carries no food-matrix occurrence data and no regulatory content.

Why this matters

• It is one of the earliest experimental demonstrations that the phytochelatin/CBP system is not sufficient to explain Cd tolerance in a unicellular green alga, contradicting the dominant 1980s view that increased phytochelatin biosynthesis is the central tolerance mechanism in plants and algae. Subsequent reviews of plant and algal metal homeostasis (Cobbett 2000, Pal & Rai 2010, Seregin & Kozhevnikova 2023, Marques 2025) cite this paper as the canonical reference for the hyperaccumulator-style observation that high-tolerance phenotypes can coexist with lower phytochelatin-bound Cd fractions. It is the empirical predecessor to the modern consensus, articulated in Phytochelatins: Sulfur-Containing Metal(loid)-Chelating Ligands in Plants, that phytochelatins are involved in metal homeostasis but not in hyperaccumulation.
• It anchors a quantitative comparison the wiki’s mitigation-evidence section may cite: 50% growth inhibition (EC50) of CW-15 occurred at ~35 μM Cd; CW15-Cdr at ~110 μM Cd — a ~3-fold tolerance gain achieved by long-term selection over ~9 months in batch culture under continuously increased Cd concentrations. The tolerant cells survive 300 μM Cd and grow stably in 60 μM Cd for ≥150 cell cycles. These are the dose anchors any downstream review citing this paper relies on.
• It corroborates a Silene cucubalus observation from the same era (cited as reference 6 in the paper: Grill, Winnacker & Zenk 1988 Experientia 44:539–540) that Cd-tolerant Silene cucubalus plants growing on heavy-metal-contaminated soils contain low amounts of phytochelatins. The two observations, one in higher plants and one in an algal model, jointly grounded the late-1980s shift in the field’s mechanistic narrative.
• It documents the chloroplast-side phenotype of the tolerant strain: the tolerant strain’s basal chlorophyll (7.2 ± 1.6 pg/cell) is 60% of the sensitive strain’s value (11.9 ± 2.7 pg/cell) — i.e., a 40% reduction in baseline chlorophyll; the Cd-induced chlorophyll loss at 60 μM Cd is 25% in CW15-Cdr versus 85% in CW-15; and there is a compensatory increase in starch accumulation in the absence of Cd (3.6× over 12 h in the tolerant strain vs 2.1× in the sensitive strain). The authors propose this is consistent with a mutation in a photosystem II component that simultaneously reduces baseline photosynthetic capacity and reduces the photosystem-II-specific Cd-sensitivity of the tolerant cells. This is the chloroplast-pathway tolerance hypothesis that subsequent papers from the same Hamburg lab (Voigt & Nagel 1995 Microbiol. Res., Voigt & Nagel 1998 J. Plant Physiol.) developed.

Key concepts and structure

The paper is a three-page Notes article (pages 526–528 of Volume 55, Number 2 of Applied and Environmental Microbiology, February 1989). It is structured as a short narrative without explicit section headings other than a one-paragraph abstract and a literature-cited block. The text flows from the toxicological context (Cd as an environmental pollutant; the role of metallothioneins in animals and phytochelatins in plants), through the selection protocol (long-term batch culture of CW-15 under continuously increased Cd over ~9 months), through the dose-response characterisation (cell growth and chlorophyll content versus CdCl2 concentration; Figure 1), to the radiotracer experiment (109Cd uptake and intracellular distribution; Table 1), to the chlorophyll accumulation kinetics under Cd (Figure 2), to the starch quantification (Figure 3), and finally to the discussion of the metabolic-versus-detoxification interpretation.

Strain origin and selection protocol

CW-15 is a cell-wall-deficient mutant of Chlamydomonas reinhardtii derived as described by Davies & Plaskitt (1971) Genet. Res. Camb. 17:33–43 (cited as reference 3). The cell-wall deficiency facilitates Cd entry because intracellular accumulation in cell-walled algae is partly limited by binding to the wall. Long-term selection was performed by culturing CW-15 in axenic batch culture under mixotrophic conditions in CTM (cadmium test medium, composition: 0.5 mM MgCl2, 0.5 mM CaCl2, 7.5 mM NH4H2PO4, 7.5 mM KCl, 5 mM PIPES at pH 6.3, with 2% sodium acetate and trace elements per Starr 1971) at increasing CdCl2 concentrations over ~9 months. The resulting population is designated CW15-Cdr.

Dose response (Figure 1)

In CTM medium, 50% growth inhibition of the parent CW-15 occurred at ~35 μM Cd (24h exposure); CW-15 cells died at ~70 μM Cd. The tolerant CW15-Cdr population showed 50% growth inhibition at ~110 μM Cd; many tolerant cells survived 300 μM Cd at 72 h. CW15-Cdr was maintained continuously in 60 μM Cd for ≥150 cell cycles with no observed variation in growth. Cells were synchronised by 12 h light–12 h dark cycling at 15 ± 2°C. Cell densities and chlorophyll were measured at 48 h, with starting density 2 × 10^5 to 2.5 × 10^5 cells per ml; the relative growth and chlorophyll values were normalised to the no-Cd control of each strain.

109Cd uptake and intracellular distribution (Table 1)

After 48 h in 30 μM CdCl2 with 0.25 MBq of 109CdCl2, cells were sonicated and the cytosolic Cd-binding components were separated by gel filtration into three molecular-weight classes (high-molecular-weight, CBP-fraction phytochelatins, and low-molecular-weight). Values are means ± SD; numbers in parentheses are percent of total uptake.

Total Cd uptake was higher in the tolerant strain (220 ± 25 vs 190 ± 25 kcpm/10^9 cells); the CBP-bound fraction was lower in absolute terms (25,000 ± 5,000 vs 45,000 ± 3,750 cpm/10^9 cells) and substantially lower in percentage terms (39% vs 67% of cytosolic Cd-binding signal). High-molecular-weight complexes and low-molecular-weight complexes both rose in the tolerant strain (HMW: 11,000 ± 4,500 vs 4,300 ± 1,000; LMW: 21,000 ± 4,000 vs 14,000 ± 3,000). The authors interpret this as evidence that the CBP system is not load-bearing for tolerance in CW15-Cdr.

Chlorophyll accumulation kinetics (Figure 2)

The CW-15 parent’s basal chlorophyll was 11.9 ± 2.7 pg/cell (no Cd); the CW15-Cdr derivative had 7.2 ± 1.6 pg/cell — the tolerant strain’s basal chlorophyll is 60% of the sensitive strain’s value (i.e., a 40% reduction). Figure 2 of the source shows relative chlorophyll trajectories (normalised) under 0, 30, and 60 μM Cd in both strains across a 48 h time course: at 30 μM Cd, the CW-15 trajectory is strongly suppressed while the CW15-Cdr trajectory continues to climb; at 60 μM Cd, CW-15 chlorophyll content fell 85% while CW15-Cdr fell only 25%. The chlorophyll deficit is consistent with the authors’ proposal that the mutation is in a photosystem II component that simultaneously reduces baseline photosynthesis and reduces Cd-sensitivity of the photosystem.

Starch content (Figure 3)

Starch content was measured enzymatically at the beginning and end of a 12 h light period. The Figure 3 conditions reported are CW-15 (no Cd), CW-15 (+30 μM Cd), CW15-Cdr (no Cd), and CW15-Cdr (+100 μM Cd); the source did not test CW-15 at 100 μM Cd because CW-15 dies at ~70 μM Cd. Fold-increase over 12 h of illumination, per the source text (p. 528): in the absence of Cd, CW-15 starch increased 2.1× and CW15-Cdr increased 3.6×; with Cd present, CW-15 (+30 μM Cd) starch increased 4.8× and CW15-Cdr (+100 μM Cd) increased 9.7×. The authors interpret this as the tolerant strain compensating for reduced photosystem II output by elevated starch synthesis, both at baseline and under Cd stress.

Electron microscopy

EM examination of both strains revealed no striking differences in the amount or arrangement of thylakoid membranes; the authors conclude that the chloroplast-side adaptation is biochemical (a photosystem II component mutation) rather than morphological.

Key numbers

• 50% growth-inhibition Cd concentration (EC50, 24 h, CTM medium): CW-15 parent ≈ 35 μM; CW15-Cdr tolerant ≈ 110 μM.
• Lethality threshold (CW-15): ≈ 70 μM Cd.
• Survival ceiling (CW15-Cdr): many cells survive 300 μM Cd; continuous growth in 60 μM Cd for ≥150 cell cycles.
• Selection duration: ~9 months of batch-culture passaging.
• Radiotracer dose: 30 μM CdCl2 + 0.25 MBq 109CdCl2; 48 h incubation; measurement at the end of incubation.
• Total 109Cd uptake: CW-15 = 190 ± 25 kcpm/10^9 cells; CW15-Cdr = 220 ± 25 kcpm/10^9 cells.
• CBP-bound Cd (cytosolic fraction): CW-15 = 45,000 ± 3,750 cpm/10^9 cells (67 ± 6%); CW15-Cdr = 25,000 ± 5,000 cpm/10^9 cells (39 ± 8%).
• Basal chlorophyll: CW-15 = 11.9 ± 2.7 pg/cell; CW15-Cdr = 7.2 ± 1.6 pg/cell (60% of the CW-15 value, i.e., a 40% reduction).
• Cd-induced chlorophyll loss at 60 μM Cd: CW-15 = 85%; CW15-Cdr = 25%.
• Starch fold-increase over 12 h of light (per source text, p. 528; Fig. 3 reports four conditions only): CW-15 no Cd = 2.1×; CW15-Cdr no Cd = 3.6×; CW-15 + 30 μM Cd = 4.8×; CW15-Cdr + 100 μM Cd = 9.7×. CW-15 was not tested at 100 μM Cd (above its lethality threshold of ~70 μM); CW15-Cdr was not separately reported at 30 μM Cd.

Methods (brief)

Strains: Chlamydomonas reinhardtii CW-15 (cell-wall-deficient mutant; Davies & Plaskitt 1971) and CW15-Cdr (Cd-tolerant population derived in this study by ~9 months of selection in CTM medium with continuously increased CdCl2 concentrations). Reference strains: W 11-32c (wild-type) and W 137c (mutant strain) included in Figure 1 dose-response curves for comparison.

Cultures: axenic batch culture under mixotrophic conditions (CTM medium: 0.5 mM MgCl2, 0.5 mM CaCl2, 7.5 mM NH4H2PO4, 7.5 mM KCl, 5 mM PIPES at pH 6.3, 2% sodium acetate, trace elements per Starr 1971). Light-dark cycling for synchronisation: 12 h light, 12 h dark per Surzycki (1971) Methods Enzymol. 23:67–73 and Voigt & Münzner (1987) Planta 172:463–472 (no temperature reported in the present paper). Starting density for dose-response: 2 × 10^5 to 2.5 × 10^5 cells/ml; 48 h incubation. Cell density by duplicate haemocytometer counts. Chlorophyll by Arnon (1949) Plant Physiol. 24:1–15.

Cd quantification: 109Cd radiolabel, filtered on Whatman GF-C at 800 ± 20 hPa. CBP isolation by sonication in extraction buffer of Rauser (1987) Experientia Suppl. 52:301–308 with chromatographic separation and classification per Hart & Bertram (1980) Environ. Exp. Bot. 20:175–180. Protein by Lowry et al. (1951) J. Biol. Chem. 193:265–275 and Bradford (1976) Anal. Biochem. 72:248–254. Starch by Boehringer (Mannheim, Germany) enzymatic test kit.

Electron microscopy: technical assistance by I. Wachholz, E. Manshard, and M. Mix (unspecified protocol; thylakoid membrane comparison only). uranium (U). Adelmeier is acknowledged for “stimulating discussion”, not for the EM work itself.

Statistics: values reported as mean ± SD. No formal statistical comparison between strains is reported; the authors rely on the magnitude of differences and on the consistency of the pattern (uptake higher, CBP-bound lower) for inference.

Funded by Deutsche Forschungsgemeinschaft grant Na 146/1.

Implications

• Microbiome: Not relevant. Chlamydomonas reinhardtii is not a gut-microbiome organism; no microbiome implications.

Limitations

• The radiotracer experiment is a single 48-hour endpoint with no time-course data, no replicate-experiment count reported beyond the SD on the mean, and no formal between-strain statistical test. The strength of the conclusion (“CBP do not play the central role in Cd tolerance”) rests on the magnitude of the percent-bound difference (67% vs 39%) and on the consistency of the pattern (the tolerant strain takes up more Cd but binds less of it to CBP), not on a formal statistical comparison.
• The molecular identity of the photosystem II component proposed to be mutated is not established here; the authors propose the hypothesis but do not isolate or sequence any candidate gene. Subsequent papers from the same Hamburg lab (Voigt & Nagel 1995, Voigt & Nagel 1998 — both indexed in the same citation cluster but not in the wiki corpus as of this ingest) developed the photosystem II component characterisation further.
• The CW-15 parent is a cell-wall-deficient mutant, which makes the model unrepresentative of wild-type Chlamydomonas reinhardtii (let alone of cell-walled plant cells in food crops). Generalisation to plant cells in agricultural settings requires bridging assumptions the paper does not test.
• Sample size is not reported in cell or population terms; values are means ± SD across an unspecified number of biological or technical replicates. The “n=” descriptor cannot be filled from the paper text.
• The 1989 selection protocol is descriptive (cells were “kept in medium containing 60 μM Cd for more than 150 cell cycles”) rather than rigorously controlled (no parallel passage control, no genome sequencing, no clonality verification). The “Cd-tolerant population” is a population-level phenotype, not necessarily a single genetic lineage; the authors acknowledge they cannot rule out reversion to the cell-walled phenotype as a confounder (they checked by electron microscopy and ruled out cell-wall reversion).
• The CBP fraction is operationally defined by chromatographic elution position per Hart & Bertram (1980); the paper does not directly demonstrate that the fraction comprises phytochelatins as we now understand them (the Grill, Winnacker & Zenk identification of phytochelatins in higher plants was 1985–1988; the Grill 1989 PNAS characterisation of phytochelatin synthase enzymology was published 6 months after this Nagel & Voigt paper). The “CBP” terminology in 1989 covered a broader and less precisely defined class of cytosolic Cd-binding components than the modern phytochelatin definition.
• No occurrence data; no consumer exposure data; no regulatory implication.

Related evidence

• Remediation evidence — drivers and interventions
• Peptides Used for Heavy Metal Remediation: A Promising Approach
• Phytochelatins and Cadmium Mitigation: Harnessing Genetic Avenues for Plant Functional Manipulation

Update history

The five most recent substantive edits to this page, classified major (evidence or structure moved), correction (a published value or statement was wrong and has been fixed), or minor (narrative rewritten without changing the underlying evidence). Each description is derived from what the edit did to this page; the linked commit is the authoritative record, routine regeneration passes are excluded, and the full version history lives in git.