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The Density and Length of Root Hairs Are Enhanced in Response to Cadmium and Arsenic by Modulating Gene Expressions Involved in Fate Determination and Morphogenesis of Root Hairs in Arabidopsis

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ABSTRACT

Root hairs are tubular outgrowths that originate from epidermal cells. Exposure of Arabidopsis to cadmium (Cd) and arsenic [arsenite, As(III)] increases root hair density and length. To examine the underlying mechanism, we measured the expression of genes involved in fate determination and morphogenesis of root hairs. Cd and As(III) downregulated TTG1 and GL2 (negative regulators of fate determination) and upregulated GEM (positive regulator), suggesting that root hair fate determination is stimulated by Cd and As(III). Cd and As(III) increased the transcript levels of genes involved in root hair initiation (RHD6 and AXR2) and root hair elongation (AUX1, AXR1, ETR1, and EIN2) except CTR1. DR5::GUS transgenic Arabidopsis showed a higher DR5 expression in the root tip, suggesting that Cd and As(III) increased the auxin content in the root tip. Knockdown of TTG1 in Arabidopsis resulted in increased root hair density and decreased root hair length compared with the control (Col-0) on 1/2 MS media. This phenotype may be attributed to the downregulation of GL2 and CTR1 and upregulation of RHD6. By contrast, gem mutant plants displayed a decrease in root hair density and length with reduced expression of RHD6, AXR2, AUX1, AXR1, ETR1, CTR1, and EIN2. Taken together, our results indicate that fate determination, initiation, and elongation of root hairs are stimulated in response to Cd and As(III) through the modulation of the expression of genes involved in these processes in Arabidopsis.

No MeSH data available.


Effect of Cd and As(III) on expressions of genes involved in the differentiation (fate determination) and elongation of root hairs in Arabidopsis.(A) Semi-quantitative RT-PCR results showing relative transcript levels of various genes involved in the differentiation (GEM, TTG1, and GL2), initiation (RHD6 and ACXR2) and elongation (AUX1, AXR1, ETR1, CTR1, and EIN2) of root hairs in response to Cd and As(III) in Arabidopsis. RT-PCR was performed using total RNA isolated from 1-week-old plants which were germinated and grown on 1/2 MS agar media treated with no metal, 40 μM CdSO4 and 10 μM As(III).The gel photography represents 3 experimental replicates. (B) Quantitative real-time PCR results of various genes involved in root hair differentiation and elongation shown in (A). Transcript levels were normalized to the transcript level of Actin.
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Figure 3: Effect of Cd and As(III) on expressions of genes involved in the differentiation (fate determination) and elongation of root hairs in Arabidopsis.(A) Semi-quantitative RT-PCR results showing relative transcript levels of various genes involved in the differentiation (GEM, TTG1, and GL2), initiation (RHD6 and ACXR2) and elongation (AUX1, AXR1, ETR1, CTR1, and EIN2) of root hairs in response to Cd and As(III) in Arabidopsis. RT-PCR was performed using total RNA isolated from 1-week-old plants which were germinated and grown on 1/2 MS agar media treated with no metal, 40 μM CdSO4 and 10 μM As(III).The gel photography represents 3 experimental replicates. (B) Quantitative real-time PCR results of various genes involved in root hair differentiation and elongation shown in (A). Transcript levels were normalized to the transcript level of Actin.

Mentions: Our findings that exposure to metals increased root hair density and length suggested that Cd and As(III) promoted the differentiation and elongation of root hairs. Developmental pathways regulated by the TTG1/GL2 interaction determine root epidermal cell fate (Masucci and Schiefelbein, 1996). We therefore measured the expression levels of TTG1 and GL2 using semi-quantitative RT-PCR (Figure 3A, graphs in Supplementary Figure S1), qRT-PCR (Figure 3B), and GUS staining (Figures 4A,B). Cd and As(III) markedly downregulated TTG1 expression compared with that in the control, providing an explanation for the increment in root hair density induced by Cd and As(III), since TTG1 is a negative regulator of root hair differentiation (Figures 3A,B). The expression of GL2, another negative regulator of root hair differentiation, was also repressed by Cd and As(III). In addition, GUS activity in Arabidopsis plants expressing the GL2::GUS construct which were germinated and grown for 5 days on medium containing Cd and As(III) decreased compared with that in controls, suggesting that GL2 expression is downregulated by Cd and As(III) (Figures 4A,B). This finding is contrary to previous studies showing that GL2-GUS is preferentially expressed in developing atrichoblast cells within the meristematic and elongation zones of the Arabidopsis root (Masucci et al., 1996). Taken together, these results indicated that the Cd or As(III) induced increase in root hair density was probably caused by the inhibition of GL2 and TTG1 gene expression. Because GEM plays a positive role in root hair fate determination (Caro et al., 2007), we examined the expression of GEM in Cd and As(III) treated plants. We found that the two metals significantly increased GEM mRNA levels compared with those in control untreated plants (Figures 3A,B). Taken together, our results suggested that Cd and As(III) increased root hair density by promoting root hair differentiation through the repression of TTG1 and GL2 (negative regulators of root hair differentiation) and the upregulation of GEM expression (positive regulator).


The Density and Length of Root Hairs Are Enhanced in Response to Cadmium and Arsenic by Modulating Gene Expressions Involved in Fate Determination and Morphogenesis of Root Hairs in Arabidopsis
Effect of Cd and As(III) on expressions of genes involved in the differentiation (fate determination) and elongation of root hairs in Arabidopsis.(A) Semi-quantitative RT-PCR results showing relative transcript levels of various genes involved in the differentiation (GEM, TTG1, and GL2), initiation (RHD6 and ACXR2) and elongation (AUX1, AXR1, ETR1, CTR1, and EIN2) of root hairs in response to Cd and As(III) in Arabidopsis. RT-PCR was performed using total RNA isolated from 1-week-old plants which were germinated and grown on 1/2 MS agar media treated with no metal, 40 μM CdSO4 and 10 μM As(III).The gel photography represents 3 experimental replicates. (B) Quantitative real-time PCR results of various genes involved in root hair differentiation and elongation shown in (A). Transcript levels were normalized to the transcript level of Actin.
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Figure 3: Effect of Cd and As(III) on expressions of genes involved in the differentiation (fate determination) and elongation of root hairs in Arabidopsis.(A) Semi-quantitative RT-PCR results showing relative transcript levels of various genes involved in the differentiation (GEM, TTG1, and GL2), initiation (RHD6 and ACXR2) and elongation (AUX1, AXR1, ETR1, CTR1, and EIN2) of root hairs in response to Cd and As(III) in Arabidopsis. RT-PCR was performed using total RNA isolated from 1-week-old plants which were germinated and grown on 1/2 MS agar media treated with no metal, 40 μM CdSO4 and 10 μM As(III).The gel photography represents 3 experimental replicates. (B) Quantitative real-time PCR results of various genes involved in root hair differentiation and elongation shown in (A). Transcript levels were normalized to the transcript level of Actin.
Mentions: Our findings that exposure to metals increased root hair density and length suggested that Cd and As(III) promoted the differentiation and elongation of root hairs. Developmental pathways regulated by the TTG1/GL2 interaction determine root epidermal cell fate (Masucci and Schiefelbein, 1996). We therefore measured the expression levels of TTG1 and GL2 using semi-quantitative RT-PCR (Figure 3A, graphs in Supplementary Figure S1), qRT-PCR (Figure 3B), and GUS staining (Figures 4A,B). Cd and As(III) markedly downregulated TTG1 expression compared with that in the control, providing an explanation for the increment in root hair density induced by Cd and As(III), since TTG1 is a negative regulator of root hair differentiation (Figures 3A,B). The expression of GL2, another negative regulator of root hair differentiation, was also repressed by Cd and As(III). In addition, GUS activity in Arabidopsis plants expressing the GL2::GUS construct which were germinated and grown for 5 days on medium containing Cd and As(III) decreased compared with that in controls, suggesting that GL2 expression is downregulated by Cd and As(III) (Figures 4A,B). This finding is contrary to previous studies showing that GL2-GUS is preferentially expressed in developing atrichoblast cells within the meristematic and elongation zones of the Arabidopsis root (Masucci et al., 1996). Taken together, these results indicated that the Cd or As(III) induced increase in root hair density was probably caused by the inhibition of GL2 and TTG1 gene expression. Because GEM plays a positive role in root hair fate determination (Caro et al., 2007), we examined the expression of GEM in Cd and As(III) treated plants. We found that the two metals significantly increased GEM mRNA levels compared with those in control untreated plants (Figures 3A,B). Taken together, our results suggested that Cd and As(III) increased root hair density by promoting root hair differentiation through the repression of TTG1 and GL2 (negative regulators of root hair differentiation) and the upregulation of GEM expression (positive regulator).

View Article: PubMed Central - PubMed

ABSTRACT

Root hairs are tubular outgrowths that originate from epidermal cells. Exposure of Arabidopsis to cadmium (Cd) and arsenic [arsenite, As(III)] increases root hair density and length. To examine the underlying mechanism, we measured the expression of genes involved in fate determination and morphogenesis of root hairs. Cd and As(III) downregulated TTG1 and GL2 (negative regulators of fate determination) and upregulated GEM (positive regulator), suggesting that root hair fate determination is stimulated by Cd and As(III). Cd and As(III) increased the transcript levels of genes involved in root hair initiation (RHD6 and AXR2) and root hair elongation (AUX1, AXR1, ETR1, and EIN2) except CTR1. DR5::GUS transgenic Arabidopsis showed a higher DR5 expression in the root tip, suggesting that Cd and As(III) increased the auxin content in the root tip. Knockdown of TTG1 in Arabidopsis resulted in increased root hair density and decreased root hair length compared with the control (Col-0) on 1/2 MS media. This phenotype may be attributed to the downregulation of GL2 and CTR1 and upregulation of RHD6. By contrast, gem mutant plants displayed a decrease in root hair density and length with reduced expression of RHD6, AXR2, AUX1, AXR1, ETR1, CTR1, and EIN2. Taken together, our results indicate that fate determination, initiation, and elongation of root hairs are stimulated in response to Cd and As(III) through the modulation of the expression of genes involved in these processes in Arabidopsis.

No MeSH data available.