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Lead tolerance and accumulation in Hirschfeldia incana, a Mediterranean Brassicaceae from metalliferous mine spoils.

Auguy F, Fahr M, Moulin P, Brugel A, Laplaze L, Mzibri ME, Filali-Maltouf A, Doumas P, Smouni A - PLoS ONE (2013)

Bottom Line: The functional characterization of HiHMA4 and HiMT2a was achieved using Arabidopsis T-DNA insertional mutants.Pb content and primary root growth analysis confirmed the role of these two genes in Pb tolerance and accumulation.H. incana could be considered as a good experimental model to identify genes involved in lead tolerance and accumulation in plants.

View Article: PubMed Central - PubMed

Affiliation: Institut de Recherche pour le Développement, Unité Mixte de Recherche Diversité Adaptation et Développement des Plantes, Montpellier, France.

ABSTRACT
Lead is a heavy metal of particular concern with respect to environmental quality and health. The lack of plant species that accumulate and tolerate Pb is a limiting factor to understand the molecular mechanisms involved in Pb tolerance. In this study we identified Hirschfeldia incana, a Brassicaceae collected from metalliferous mine spoils in Morocco, as a Pb accumulator plant. H. incana exhibited high Pb accumulation in mine soils and in hydroponic cultures. Major Pb accumulation occurred in the roots and a part of Pb translocated from the roots to the shoots, even to the siliques. These findings demonstrated that H. incana is a Pb accumulator species. The expression of several candidate genes after Pb-exposure was measured by quantitative PCR and two of them, HiHMA4 and HiMT2a, coding respectively for a P1B-type ATPase and a metallothionein, were particularly induced by Pb-exposure in both roots and leaves. The functional characterization of HiHMA4 and HiMT2a was achieved using Arabidopsis T-DNA insertional mutants. Pb content and primary root growth analysis confirmed the role of these two genes in Pb tolerance and accumulation. H. incana could be considered as a good experimental model to identify genes involved in lead tolerance and accumulation in plants.

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Effects of lead treatment on primary root length in wild-type and knockout mutant seedlings of A. thaliana.Seedlings were grown for 13 days on vertically orientated agar plates with or without 40 µM of Pb(NO3)2. (A) Atmt2a T-DNA mutant analysis. (B) Athma4 T-DNA mutant analysis. All results are the average value (± SE) of 16 seedlings. The letters represent statistically homogenous subgroups using LSD post hoc test at a α = 0.01 significance level.
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pone-0061932-g006: Effects of lead treatment on primary root length in wild-type and knockout mutant seedlings of A. thaliana.Seedlings were grown for 13 days on vertically orientated agar plates with or without 40 µM of Pb(NO3)2. (A) Atmt2a T-DNA mutant analysis. (B) Athma4 T-DNA mutant analysis. All results are the average value (± SE) of 16 seedlings. The letters represent statistically homogenous subgroups using LSD post hoc test at a α = 0.01 significance level.

Mentions: If HiHMA4 and HiMT2a are important for lead tolerance, as suggested by their overexpression in the presence of Pb, we expect the corresponding Arabidopsis T-DNA mutants to be more sensitive to heavy metals than wild type plants. To test the physiological function of HMA4 and MT2a in A. thaliana, two homozygote lines that contain T-DNA insertion in the HMA4 gene (Salk_093482, Figure S3) and in the MT2a gene (Salk_059712, Figure S4) respectively were isolated from the T-DNA insertion collection generated at the Salk Institute (http://signal.salk.edu). To evaluate the consequences of HMA4 and MT2a gene disruption on root growth responses to Pb treatment, we examined the primary root length of mutant plants grown under Pb-treatment and control conditions and found them to be distinguishable from the wild type plants. Wild-type, hma4 and mt2a seeds were germinated directly on standard medium or on medium with 40 µM Pb(NO3)2. The lead concentration used in this experiment corresponds to the threshold of tolerance that Arabidopsis can withstand in our experimental conditions (data not shown). After 13 days of culture, Pb-treated wild-type plants did not seem to be affected by the Pb treatment compared to the control plants. Similarly when grown on standard medium, both hma4 and mt2a mutants and wild-type plants showed a similar root phenotype. Pb treatment highly significantly reduced primary root length in both hma4 (45%) and in mt2a (48%) mutants (Figure 6). This reduction in primary root growth reflects an increased sensitivity to Pb in the Arabidopsis T-DNA insertion mutants and it suggests that the HMA4 and MT2a genes contribute to lead tolerance. We measured the Pb contents in roots and shoots of the 2 week-old wild-type and hma4- and mt2a-mutants (Figure 7). Roots of hma4 plants contained 1.8-fold more Pb than those of the wild-type plants (p<0.01). Although the Pb content of mt2a-plants seems slightly higher than that of wild-type plants in Pb(NO3)2-containing medium, the difference was not statistically significant. No effect was observed in Pb content in the shoots. The Pb accumulation observed in roots of hma4 plants is compatible with an interruption of the translocation resulting from HMA4 gene disruption.


Lead tolerance and accumulation in Hirschfeldia incana, a Mediterranean Brassicaceae from metalliferous mine spoils.

Auguy F, Fahr M, Moulin P, Brugel A, Laplaze L, Mzibri ME, Filali-Maltouf A, Doumas P, Smouni A - PLoS ONE (2013)

Effects of lead treatment on primary root length in wild-type and knockout mutant seedlings of A. thaliana.Seedlings were grown for 13 days on vertically orientated agar plates with or without 40 µM of Pb(NO3)2. (A) Atmt2a T-DNA mutant analysis. (B) Athma4 T-DNA mutant analysis. All results are the average value (± SE) of 16 seedlings. The letters represent statistically homogenous subgroups using LSD post hoc test at a α = 0.01 significance level.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3646990&req=5

pone-0061932-g006: Effects of lead treatment on primary root length in wild-type and knockout mutant seedlings of A. thaliana.Seedlings were grown for 13 days on vertically orientated agar plates with or without 40 µM of Pb(NO3)2. (A) Atmt2a T-DNA mutant analysis. (B) Athma4 T-DNA mutant analysis. All results are the average value (± SE) of 16 seedlings. The letters represent statistically homogenous subgroups using LSD post hoc test at a α = 0.01 significance level.
Mentions: If HiHMA4 and HiMT2a are important for lead tolerance, as suggested by their overexpression in the presence of Pb, we expect the corresponding Arabidopsis T-DNA mutants to be more sensitive to heavy metals than wild type plants. To test the physiological function of HMA4 and MT2a in A. thaliana, two homozygote lines that contain T-DNA insertion in the HMA4 gene (Salk_093482, Figure S3) and in the MT2a gene (Salk_059712, Figure S4) respectively were isolated from the T-DNA insertion collection generated at the Salk Institute (http://signal.salk.edu). To evaluate the consequences of HMA4 and MT2a gene disruption on root growth responses to Pb treatment, we examined the primary root length of mutant plants grown under Pb-treatment and control conditions and found them to be distinguishable from the wild type plants. Wild-type, hma4 and mt2a seeds were germinated directly on standard medium or on medium with 40 µM Pb(NO3)2. The lead concentration used in this experiment corresponds to the threshold of tolerance that Arabidopsis can withstand in our experimental conditions (data not shown). After 13 days of culture, Pb-treated wild-type plants did not seem to be affected by the Pb treatment compared to the control plants. Similarly when grown on standard medium, both hma4 and mt2a mutants and wild-type plants showed a similar root phenotype. Pb treatment highly significantly reduced primary root length in both hma4 (45%) and in mt2a (48%) mutants (Figure 6). This reduction in primary root growth reflects an increased sensitivity to Pb in the Arabidopsis T-DNA insertion mutants and it suggests that the HMA4 and MT2a genes contribute to lead tolerance. We measured the Pb contents in roots and shoots of the 2 week-old wild-type and hma4- and mt2a-mutants (Figure 7). Roots of hma4 plants contained 1.8-fold more Pb than those of the wild-type plants (p<0.01). Although the Pb content of mt2a-plants seems slightly higher than that of wild-type plants in Pb(NO3)2-containing medium, the difference was not statistically significant. No effect was observed in Pb content in the shoots. The Pb accumulation observed in roots of hma4 plants is compatible with an interruption of the translocation resulting from HMA4 gene disruption.

Bottom Line: The functional characterization of HiHMA4 and HiMT2a was achieved using Arabidopsis T-DNA insertional mutants.Pb content and primary root growth analysis confirmed the role of these two genes in Pb tolerance and accumulation.H. incana could be considered as a good experimental model to identify genes involved in lead tolerance and accumulation in plants.

View Article: PubMed Central - PubMed

Affiliation: Institut de Recherche pour le Développement, Unité Mixte de Recherche Diversité Adaptation et Développement des Plantes, Montpellier, France.

ABSTRACT
Lead is a heavy metal of particular concern with respect to environmental quality and health. The lack of plant species that accumulate and tolerate Pb is a limiting factor to understand the molecular mechanisms involved in Pb tolerance. In this study we identified Hirschfeldia incana, a Brassicaceae collected from metalliferous mine spoils in Morocco, as a Pb accumulator plant. H. incana exhibited high Pb accumulation in mine soils and in hydroponic cultures. Major Pb accumulation occurred in the roots and a part of Pb translocated from the roots to the shoots, even to the siliques. These findings demonstrated that H. incana is a Pb accumulator species. The expression of several candidate genes after Pb-exposure was measured by quantitative PCR and two of them, HiHMA4 and HiMT2a, coding respectively for a P1B-type ATPase and a metallothionein, were particularly induced by Pb-exposure in both roots and leaves. The functional characterization of HiHMA4 and HiMT2a was achieved using Arabidopsis T-DNA insertional mutants. Pb content and primary root growth analysis confirmed the role of these two genes in Pb tolerance and accumulation. H. incana could be considered as a good experimental model to identify genes involved in lead tolerance and accumulation in plants.

Show MeSH
Related in: MedlinePlus