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Over-expression of Arabidopsis AtCHR23 chromatin remodeling ATPase results in increased variability of growth and gene expression.

Folta A, Severing EI, Krauskopf J, van de Geest H, Verver J, Nap JP, Mlynarova L - BMC Plant Biol. (2014)

Bottom Line: Already modest (2-fold) over-expression of the AtCHR23 ATPase gene in Arabidopsis results in overall reduced growth compared to the wild-type.Detailed analyses show that in the root, the reduction of growth is due to reduced cell elongation.In contrast, the knockout mutation of AtCHR23 does not lead to such visible phenotypic effects.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Molecular Biology, Plant Sciences Group, Wageningen University and Research Centre, Droevendaalsesteeg 1, Wageningen 6708 PB, The Netherlands. ludmila.mlynarova@wur.nl.

ABSTRACT

Background: Plants are sessile organisms that deal with their -sometimes adverse- environment in well-regulated ways. Chromatin remodeling involving SWI/SNF2-type ATPases is thought to be an important epigenetic mechanism for the regulation of gene expression in different developmental programs and for integrating these programs with the response to environmental signals. In this study, we report on the role of chromatin remodeling in Arabidopsis with respect to the variability of growth and gene expression in relationship to environmental conditions.

Results: Already modest (2-fold) over-expression of the AtCHR23 ATPase gene in Arabidopsis results in overall reduced growth compared to the wild-type. Detailed analyses show that in the root, the reduction of growth is due to reduced cell elongation. The reduced-growth phenotype requires sufficient light and is magnified by applying deliberate abiotic (salt, osmotic) stress. In contrast, the knockout mutation of AtCHR23 does not lead to such visible phenotypic effects. In addition, we show that over-expression of AtCHR23 increases the variability of growth in populations of genetically identical plants. These data indicate that accurate and controlled expression of AtCHR23 contributes to the stability or robustness of growth. Detailed RNAseq analyses demonstrate that upon AtCHR23 over-expression also the variation of gene expression is increased in a subset of genes that associate with environmental stress. The larger variation of gene expression is confirmed in individual plants with the help of independent qRT-PCR analysis.

Conclusions: Over-expression of AtCHR23 gives Arabidopsis a phenotype that is markedly different from the growth arrest phenotype observed upon over-expression of AtCHR12, the paralog of AtCHR23, in response to abiotic stress. This demonstrates functional sub-specialization of highly similar ATPases in Arabidopsis. Over-expression of AtCHR23 increases the variability of growth among genetically identical individuals in a way that is consistent with increased variability of expression of a distinct subset of genes that associate with environmental stress. We propose that ATCHR23-mediated chromatin remodeling is a potential component of a buffer system in plants that protects against environmentally-induced phenotypic and transcriptional variation.

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Abiotic stress emphasizes the reduction of growth in case of AtCHR23 over-expression. (A) Photograph of 8-day-old seedlings grown at 23°C in long-day conditions on medium supplemented with 75 mM NaCl. (B) Mean (± SD) length of the primary roots of 8-day-old seedlings grown on 75 mM NaCl. (C) Photograph of 8-day-old seedlings grown at 23°C in long-day conditions on medium supplemented with 200 mM mannitol. (D) Mean (± SD) length of the primary roots of 8-day-old seedlings grown on 200 mM mannitol. (E) Photograph of 4-week-old wild-type and AtCHR23-4ov plants two weeks after application of salt stress. (F) Mean (± SD) rosette diameter of 4-week-old plants two weeks after application of salt stress. For each assay and line, 40 seedlings or 15 plants were measured. Asterisks indicate significant differences from the wild type: **, P < 0.01; ***, P < 0.001.
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Figure 6: Abiotic stress emphasizes the reduction of growth in case of AtCHR23 over-expression. (A) Photograph of 8-day-old seedlings grown at 23°C in long-day conditions on medium supplemented with 75 mM NaCl. (B) Mean (± SD) length of the primary roots of 8-day-old seedlings grown on 75 mM NaCl. (C) Photograph of 8-day-old seedlings grown at 23°C in long-day conditions on medium supplemented with 200 mM mannitol. (D) Mean (± SD) length of the primary roots of 8-day-old seedlings grown on 200 mM mannitol. (E) Photograph of 4-week-old wild-type and AtCHR23-4ov plants two weeks after application of salt stress. (F) Mean (± SD) rosette diameter of 4-week-old plants two weeks after application of salt stress. For each assay and line, 40 seedlings or 15 plants were measured. Asterisks indicate significant differences from the wild type: **, P < 0.01; ***, P < 0.001.

Mentions: The impact of modified AtCHR23 expression is also apparent in environmental stress. Seedlings were assayed under abiotic stress conditions on agar plates containing 75 mM NaCl (salt stress; Figure 6A) or 200 mM mannitol (osmotic stress; Figure 6C). Both stresses had, as expected, a clear negative impact on root growth. The average length of the roots of wild-type seedlings in an environment with salt stress was 30.92 mm (Figure 6B) and in osmotic stress 32.51 mm (Figure 6D), whereas without such stress the length was 40.7 mm (see Table 1 and Figure 1). This shows that salt stress reduces the root length of the wild-type by 24% and osmotic stress by 20%. The over-expressing mutants AtCHR23-4ov and AtCHR23-5ov respond to salt by 32% and 36% reduction of root length, respectively (Figure 6B). In osmotic stress, this reduction was 29% and 31%, respectively (Figure 6D). Similar results were obtained with the lines over-expressing AtCHR23 cDNA copy (Additional file 1: Figure S3). In contrast, the knockout line atchr23 has slightly longer roots than the wild-type, but only in osmotic stress (average length 33.9 mm; Figure 6D). These data indicate that the AtCHR23 over-expressing lines respond to stress conditions by stronger growth arrest of the root length than the wild-type. A non-parametric factor analysis showed highly significant (P < 0.001) effects of both genotype and stress treatment on root length, and significant (P < 0.01) effects of genotype X treatment interaction on root length, in all mutant lines except for knockout line at osmotic stress (Additional file 2: Table S2). The same is observed in further vegetative development. After applying salt stress by watering two-week-old plants with 100 mM NaCl twice in 3 days, the rosette diameter of soil-grown plants (Figure 6E) was measured. The rosette diameter of wild-type without stress was 34.1 mm2 whereas after stress, it was 30.34 mm2, which is a reduction of 11%. The AtCHR23-4ov plants respond to salt stress by two-fold higher (22%) reduction of the rosette diameter: from 30.1 mm2 to 23.49 mm2 (Figure 4D, 6F). The non-parametric factor analysis showed highly significant (P < 0.001) effects of both genotype and treatment on rosette diameter, however the effect of genotype X treatment interaction was not significant (Additional file 2: Table S2). It shows that abiotic stress magnifies the effect of AtCHR23 over-expression on the seedlings growth and that the effect extends beyond the seedling stage.


Over-expression of Arabidopsis AtCHR23 chromatin remodeling ATPase results in increased variability of growth and gene expression.

Folta A, Severing EI, Krauskopf J, van de Geest H, Verver J, Nap JP, Mlynarova L - BMC Plant Biol. (2014)

Abiotic stress emphasizes the reduction of growth in case of AtCHR23 over-expression. (A) Photograph of 8-day-old seedlings grown at 23°C in long-day conditions on medium supplemented with 75 mM NaCl. (B) Mean (± SD) length of the primary roots of 8-day-old seedlings grown on 75 mM NaCl. (C) Photograph of 8-day-old seedlings grown at 23°C in long-day conditions on medium supplemented with 200 mM mannitol. (D) Mean (± SD) length of the primary roots of 8-day-old seedlings grown on 200 mM mannitol. (E) Photograph of 4-week-old wild-type and AtCHR23-4ov plants two weeks after application of salt stress. (F) Mean (± SD) rosette diameter of 4-week-old plants two weeks after application of salt stress. For each assay and line, 40 seedlings or 15 plants were measured. Asterisks indicate significant differences from the wild type: **, P < 0.01; ***, P < 0.001.
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Related In: Results  -  Collection

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Figure 6: Abiotic stress emphasizes the reduction of growth in case of AtCHR23 over-expression. (A) Photograph of 8-day-old seedlings grown at 23°C in long-day conditions on medium supplemented with 75 mM NaCl. (B) Mean (± SD) length of the primary roots of 8-day-old seedlings grown on 75 mM NaCl. (C) Photograph of 8-day-old seedlings grown at 23°C in long-day conditions on medium supplemented with 200 mM mannitol. (D) Mean (± SD) length of the primary roots of 8-day-old seedlings grown on 200 mM mannitol. (E) Photograph of 4-week-old wild-type and AtCHR23-4ov plants two weeks after application of salt stress. (F) Mean (± SD) rosette diameter of 4-week-old plants two weeks after application of salt stress. For each assay and line, 40 seedlings or 15 plants were measured. Asterisks indicate significant differences from the wild type: **, P < 0.01; ***, P < 0.001.
Mentions: The impact of modified AtCHR23 expression is also apparent in environmental stress. Seedlings were assayed under abiotic stress conditions on agar plates containing 75 mM NaCl (salt stress; Figure 6A) or 200 mM mannitol (osmotic stress; Figure 6C). Both stresses had, as expected, a clear negative impact on root growth. The average length of the roots of wild-type seedlings in an environment with salt stress was 30.92 mm (Figure 6B) and in osmotic stress 32.51 mm (Figure 6D), whereas without such stress the length was 40.7 mm (see Table 1 and Figure 1). This shows that salt stress reduces the root length of the wild-type by 24% and osmotic stress by 20%. The over-expressing mutants AtCHR23-4ov and AtCHR23-5ov respond to salt by 32% and 36% reduction of root length, respectively (Figure 6B). In osmotic stress, this reduction was 29% and 31%, respectively (Figure 6D). Similar results were obtained with the lines over-expressing AtCHR23 cDNA copy (Additional file 1: Figure S3). In contrast, the knockout line atchr23 has slightly longer roots than the wild-type, but only in osmotic stress (average length 33.9 mm; Figure 6D). These data indicate that the AtCHR23 over-expressing lines respond to stress conditions by stronger growth arrest of the root length than the wild-type. A non-parametric factor analysis showed highly significant (P < 0.001) effects of both genotype and stress treatment on root length, and significant (P < 0.01) effects of genotype X treatment interaction on root length, in all mutant lines except for knockout line at osmotic stress (Additional file 2: Table S2). The same is observed in further vegetative development. After applying salt stress by watering two-week-old plants with 100 mM NaCl twice in 3 days, the rosette diameter of soil-grown plants (Figure 6E) was measured. The rosette diameter of wild-type without stress was 34.1 mm2 whereas after stress, it was 30.34 mm2, which is a reduction of 11%. The AtCHR23-4ov plants respond to salt stress by two-fold higher (22%) reduction of the rosette diameter: from 30.1 mm2 to 23.49 mm2 (Figure 4D, 6F). The non-parametric factor analysis showed highly significant (P < 0.001) effects of both genotype and treatment on rosette diameter, however the effect of genotype X treatment interaction was not significant (Additional file 2: Table S2). It shows that abiotic stress magnifies the effect of AtCHR23 over-expression on the seedlings growth and that the effect extends beyond the seedling stage.

Bottom Line: Already modest (2-fold) over-expression of the AtCHR23 ATPase gene in Arabidopsis results in overall reduced growth compared to the wild-type.Detailed analyses show that in the root, the reduction of growth is due to reduced cell elongation.In contrast, the knockout mutation of AtCHR23 does not lead to such visible phenotypic effects.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Molecular Biology, Plant Sciences Group, Wageningen University and Research Centre, Droevendaalsesteeg 1, Wageningen 6708 PB, The Netherlands. ludmila.mlynarova@wur.nl.

ABSTRACT

Background: Plants are sessile organisms that deal with their -sometimes adverse- environment in well-regulated ways. Chromatin remodeling involving SWI/SNF2-type ATPases is thought to be an important epigenetic mechanism for the regulation of gene expression in different developmental programs and for integrating these programs with the response to environmental signals. In this study, we report on the role of chromatin remodeling in Arabidopsis with respect to the variability of growth and gene expression in relationship to environmental conditions.

Results: Already modest (2-fold) over-expression of the AtCHR23 ATPase gene in Arabidopsis results in overall reduced growth compared to the wild-type. Detailed analyses show that in the root, the reduction of growth is due to reduced cell elongation. The reduced-growth phenotype requires sufficient light and is magnified by applying deliberate abiotic (salt, osmotic) stress. In contrast, the knockout mutation of AtCHR23 does not lead to such visible phenotypic effects. In addition, we show that over-expression of AtCHR23 increases the variability of growth in populations of genetically identical plants. These data indicate that accurate and controlled expression of AtCHR23 contributes to the stability or robustness of growth. Detailed RNAseq analyses demonstrate that upon AtCHR23 over-expression also the variation of gene expression is increased in a subset of genes that associate with environmental stress. The larger variation of gene expression is confirmed in individual plants with the help of independent qRT-PCR analysis.

Conclusions: Over-expression of AtCHR23 gives Arabidopsis a phenotype that is markedly different from the growth arrest phenotype observed upon over-expression of AtCHR12, the paralog of AtCHR23, in response to abiotic stress. This demonstrates functional sub-specialization of highly similar ATPases in Arabidopsis. Over-expression of AtCHR23 increases the variability of growth among genetically identical individuals in a way that is consistent with increased variability of expression of a distinct subset of genes that associate with environmental stress. We propose that ATCHR23-mediated chromatin remodeling is a potential component of a buffer system in plants that protects against environmentally-induced phenotypic and transcriptional variation.

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Related in: MedlinePlus