Understanding the development of roots exposed to contaminants and the potential of plant-associated bacteria for optimization of growth

Abstract

Background and scope: Plant responses to the toxic effects of soil contaminants, such as excess metals or organic substances, have been studied mainly at physiological, biochemical and molecular levels, but the influence on root system architecture has received little attention. Nevertheless, the precise position, morphology and extent of roots can influence contaminant uptake. Here, data are discussed that aim to increase the molecular and ecological understanding of the influence of contaminants on root system architecture. Furthermore, the potential of plant-associated bacteria to influence root growth by their growth-promoting and stress-relieving capacities is explored.

Methods: Root growth parameters of Arabidopsis thaliana seedlings grown in vertical agar plates are quantified. Mutants are used in a reverse genetics approach to identify molecular components underlying quantitative changes in root architecture after exposure to excess cadmium, copper or zinc. Plant-associated bacteria are isolated from contaminated environments, genotypically and phenotypically characterized, and used to test plant root growth improvement in the presence of contaminants.

Key results: The molecular determinants of primary root growth inhibition and effects on lateral root density by cadmium were identified. A vertical split-root system revealed local effects of cadmium and copper on root development. However, systemic effects of zinc exposure on root growth reduced both the avoidance of contaminated areas and colonization of non-contaminated areas. The potential for growth promotion and contaminant degradation of plant-associated bacteria was demonstrated by improved root growth of inoculated plants exposed to 2,4-di-nitro-toluene (DNT) or cadmium.

Conclusions: Knowledge concerning the specific influence of different contaminants on root system architecture and the molecular mechanisms by which this is achieved can be combined with the exploitation of plant-associated bacteria to influence root development and increase plant stress tolerance, which should lead to more optimal root systems for application in phytoremediation or safer biomass production.

Fig. 1.

Primary root growth of A. thaliana wild-type and lox1-1 mutant seedlings (as indicated) during 7 d exposure on vertical agar plates to CdSO₄ (A) or CuSO₄ (B), expressed relative to the control (0 µm) for each genotype. Significant genotype effects within treatments are indicated (*P < 0·05, t-test; n = 20).

Fig. 2.

(A) Seven-day-old A. thaliana plants, with primary root length equal to the white mark, were exposed for another 7 d to 5 µm CdSO₄, 10 µm CuSO₄ or 75 µm ZnSO₄. At a similar inhibition of primary root growth, lateral root density was increased by Cd and Cu, but decreased by Zn exposure. Lateral root elongation was less affected by Cu than by Cd. The effect on lateral root density and elongation may affect the ability of the lateral root tips to reach non-contaminated zones, for which the response to Cu may be more optimal. (Note: image contrast was enhanced to visualize roots, and shoot colour may look artificial). (B) Lateral root density (base to apex) of A. thaliana wild-type and ein3-1 mutant seedlings (as indicated) after 7 d exposure on vertical agar plates to CdSO₄. Significant genotype effects within treatments are indicated (*P < 0·05, non-parametric Kruskal–Wallis test, n = 6).

Fig. 3.

(A) In the vertical split root-system, 8-d-old seedlings grown under control conditions were positioned such that the primary root tip was exposed to a different treatment from the rest of the root system (control conditions have the same treatment in the upper and lower zone). Visualized here are plants exposed at the growing root tip only to 5 µm CdSO₄, 10 µm CuSO₄ or 75 µm ZnSO₄. Lateral root growth in the upper non-contaminated zone is visually compromised by Zn exposure of the primary root tip, which is not the case for plants exposed to Cd or Cu at the primary root tip. (B) Primary root growth rates of A. thaliana wild-type seedlings 7 d after transfer of the seedlings to vertical split-root plates containing heterogeneous conditions of CdSO₄, CuSO₄ or ZnSO₄. Indicated in the legend is the metal concentration (μm) in the top zone and the concentration in the bottom zone (n = 6–14).

Fig. 4.

Primary root length (A), total lateral root (LR) length (B), number of laterals (C) and mean lateral root length (D) 7 d after transfer of 8-d-old A. thaliana seedlings to vertical split-root plates, in which the primary root tip was positioned in the bottom zone and the rest of the root system in the top zone. The x-axis values indicate the exposure concentrations (μm) as ‘top zone–bottom zone’. Plants were exposed to CdSO₄, CuSO₄ or ZnSO₄, colour-coded on the figure; black indicates zones with zero exposure. Different letters indicate statistically significant differences (P < 0·05) after one-way ANOVA and Tukey correction (n = 6–14) (ng, no growth detected).

Fig. 5.

Inoculation with seed endophytes can enhance root growth under Cd stress. Arabidopsis thaliana control seeds (from plants that were never exposed to Cd) as well as Cd seeds (from plants that were exposed to 2 µm Cd for several generations) were grown on vertical agar plates containing 0, 2 or 10 µm CdSO₄ during the entire experiment. Half of the 7-d-old control plants were inoculated by transferring them to plates on which a bacterial suspension was streaked out (108 c.f.u. ml⁻¹) representing the endophyte population isolated from Cd seeds (see text for species composition). (A, B) Primary root length of control plants, Cd plants and inoculated control plants after 21 d exposure to (A) 2 µm Cd or (B) 10 µm Cd (***P < 0·001, one-way ANOVA with Tukey correction, n = 20). (C, D) Primary root growth rate for control plants, Cd plants and inoculated plants upon exposure to (C) 2 or (D) 10 µm Cd. Day 1 on the graphs corresponds to the first day after inoculation (*P < 0·05, **P < 0·01, ***P < 0·001 within-day one-way ANOVA with Tukey correction, n = 20). (E) Vertical agar plate with a control plant (a), Cd plant (b) and inoculated plant (c).

Fig. 6.

Bacteria isolated from an explosives-polluted soil are capable of enhancing root growth in presence of 2,4-DNT contamination. (A) 2,4-DNT inhibits primary root growth when 7-d- old A. thaliana seedlings are grown over 9 d on plates amended with different 2,4-DNT concentrations (0 – 10 mg L⁻¹, data are mean ± s.e.; ***P < 0·001, non-parametric Kruskal–Wallis test; n = 12). (B) Arabidopsis thaliana growth on 0 mg L⁻¹ (a) and 1 mg L⁻¹ (b) 2,4-DNT. (C) Primary root length 9 d after transfer of 7-d-old seedlings in response to 2,4-DNT (0 and 1 mg L⁻¹) and in the presence or absence of bacteria tested for 2,4-DNT degradation and plant-growth promoting characteristics (IAA production and ACC-deaminase activity, see text). 10³ c.f.u. was spread on a plate to inoculate the transferred seedlings (data are mean ± s.e.; ***P < 0·001, **P < 0·01, non-parametric Kruskal–Wallis test; n = 12). (D) Visualization of root hair formation by staining with Crystal Violet in 15-d-old A. thaliana seedlings exposed to 1 mg L⁻¹ DNT and in the presence or absence of bacteria. (a) Non-inoculated control, (b) Burkholderia, (c) Sphingomonas sp., (d) Bacillus sp., (e) Ralstonia sp., (f) Geobacillus sp.