Premise of the study: Plants grown at high densities show increased tolerance to heavy metals for reasons that are not clear. A potential explanation is the release of citrate by plant roots, which binds metals and prevents uptake. Thus, pooled exudates at high plant densities could increase tolerance. We tested this exclusion facilitation hypothesis using mutants of Arabidopsis thaliana defective in citrate exudation.

Methods: Wild type Arabidopsis and two allelic mutants for the Ferric Reductase Defective 3 (FRD3) gene were grown at four densities and watered with copper sulfate at four concentrations. Plants were harvested before bolting and dried. Shoot biomass was measured, and shoot material and soil were digested in nitric acid. Copper contents were determined by atomic absorption.

Key results: In the highest copper treatment, density-dependent reduction in toxicity was observed in the wild type but not in FRD3 mutants. For both mutants, copper concentrations per gram biomass were up to seven times higher thanwild type plants, depending on density and copper treatment. In all genotypes, total copper accumulation was greater at higher plant densities. Plant size variation increased with density and copper treatment because of heterogeneous distribution of copper throughout the soil.

Conclusions: These results support the hypothesis that citrate exudation is responsible for density-dependent reductions in toxicity of metals. Density-dependent copper uptake and growth in contaminated soils underscores the importance of density in ecotoxicological testing. In soils with a heterogenous distribution of contaminants, competition for non-toxic soil regions may drive size hierarchies and determine competitive outcomes. 

Biomass is for dry weight at harvest; copper concentrations were determined by atomic absorption following acid digestion of shoots.