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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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Box plots of the relative mRNA levels of selected genes in individual seedlings. Relative mRNA levels were determined in six individual seedlings, each of wild-type (Col) and three AtCHR23 over-expressing lines, by quantitative RT-PCR using gene specific primers for eight different genes. Four of these genes (A) were characterized by high variability between the replicates of the over-expressing lines and four genes (B) did not show such variability. After normalization to the UBC reference gene, the expression value for each gene and each individual seedling was scaled relative to the lowest expression value (set to 1) for that gene in the line. In each box plot, the box area represents the lower and the upper percentiles, the horizontal line within the box indicates the median value and the horizontal dashes at the top and the bottom of the box show the minimum and maximum values observed. Asterisk indicate significantly different variances (Levene’s test) from the wild type at P < 0.05.
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Figure 10: Box plots of the relative mRNA levels of selected genes in individual seedlings. Relative mRNA levels were determined in six individual seedlings, each of wild-type (Col) and three AtCHR23 over-expressing lines, by quantitative RT-PCR using gene specific primers for eight different genes. Four of these genes (A) were characterized by high variability between the replicates of the over-expressing lines and four genes (B) did not show such variability. After normalization to the UBC reference gene, the expression value for each gene and each individual seedling was scaled relative to the lowest expression value (set to 1) for that gene in the line. In each box plot, the box area represents the lower and the upper percentiles, the horizontal line within the box indicates the median value and the horizontal dashes at the top and the bottom of the box show the minimum and maximum values observed. Asterisk indicate significantly different variances (Levene’s test) from the wild type at P < 0.05.

Mentions: To address the impact of variation on gene expression in individual seedlings, eight genes were selected for additional analyses. Four genes were randomly selected from the list of AtCHR23-4ov variable genes at long-day conditions. In addition four genes were randomly selected that were identified as not variable (including AtCHR23/At5g19310). Details of these genes are given in Additional file 2: Table S3. The expression of these eight genes was analyzed by quantitative RT-PCR in six individual seedlings of over-expressing mutants and the wild-type grown at long-day conditions. Box plots summarizing these data show considerably more variation in expression among individual seedlings of the various mutants compared to the wild-type for the four variable genes (Figure 10A). In contrast, none of the genes selected for lack of variation showed such a large expression variability between individual seedlings in any of line tested (Figure 10B). In individual seedlings different from seedlings analyzed in Figure 10 additional three variable (At1g04220, At3g22640, At3g12580) and three not variable genes (At5g02490, At5g10140, At2g01422) were analyzed. For all of them, except for At3g12580, the variability as detected by RNAseq was confirmed. Although based on limited number of individuals these data show that the increased variation of gene expression of distinct subset of genes is also apparent in individual seedlings. This emphasizes the importance of studying the expression pattern in individual plants. For direct biological proof of increased expression variation in AtCHR23 over-expressing mutant more genes should be tested, preferably by extensive RNAseq analysis of a larger number of individual seedlings.


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)

Box plots of the relative mRNA levels of selected genes in individual seedlings. Relative mRNA levels were determined in six individual seedlings, each of wild-type (Col) and three AtCHR23 over-expressing lines, by quantitative RT-PCR using gene specific primers for eight different genes. Four of these genes (A) were characterized by high variability between the replicates of the over-expressing lines and four genes (B) did not show such variability. After normalization to the UBC reference gene, the expression value for each gene and each individual seedling was scaled relative to the lowest expression value (set to 1) for that gene in the line. In each box plot, the box area represents the lower and the upper percentiles, the horizontal line within the box indicates the median value and the horizontal dashes at the top and the bottom of the box show the minimum and maximum values observed. Asterisk indicate significantly different variances (Levene’s test) from the wild type at P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3987066&req=5

Figure 10: Box plots of the relative mRNA levels of selected genes in individual seedlings. Relative mRNA levels were determined in six individual seedlings, each of wild-type (Col) and three AtCHR23 over-expressing lines, by quantitative RT-PCR using gene specific primers for eight different genes. Four of these genes (A) were characterized by high variability between the replicates of the over-expressing lines and four genes (B) did not show such variability. After normalization to the UBC reference gene, the expression value for each gene and each individual seedling was scaled relative to the lowest expression value (set to 1) for that gene in the line. In each box plot, the box area represents the lower and the upper percentiles, the horizontal line within the box indicates the median value and the horizontal dashes at the top and the bottom of the box show the minimum and maximum values observed. Asterisk indicate significantly different variances (Levene’s test) from the wild type at P < 0.05.
Mentions: To address the impact of variation on gene expression in individual seedlings, eight genes were selected for additional analyses. Four genes were randomly selected from the list of AtCHR23-4ov variable genes at long-day conditions. In addition four genes were randomly selected that were identified as not variable (including AtCHR23/At5g19310). Details of these genes are given in Additional file 2: Table S3. The expression of these eight genes was analyzed by quantitative RT-PCR in six individual seedlings of over-expressing mutants and the wild-type grown at long-day conditions. Box plots summarizing these data show considerably more variation in expression among individual seedlings of the various mutants compared to the wild-type for the four variable genes (Figure 10A). In contrast, none of the genes selected for lack of variation showed such a large expression variability between individual seedlings in any of line tested (Figure 10B). In individual seedlings different from seedlings analyzed in Figure 10 additional three variable (At1g04220, At3g22640, At3g12580) and three not variable genes (At5g02490, At5g10140, At2g01422) were analyzed. For all of them, except for At3g12580, the variability as detected by RNAseq was confirmed. Although based on limited number of individuals these data show that the increased variation of gene expression of distinct subset of genes is also apparent in individual seedlings. This emphasizes the importance of studying the expression pattern in individual plants. For direct biological proof of increased expression variation in AtCHR23 over-expressing mutant more genes should be tested, preferably by extensive RNAseq analysis of a larger number of individual seedlings.

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.

Show MeSH
Related in: MedlinePlus