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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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Scatter plots of gene expression expressed as log2(FPKM + 1) show more pronounced variability in long-day grown over-expressing mutant. Expression was determined from RNAseq reads for the wild-type (Columbia) and mutant (AtCHR23-4ov), with biological replicates indicated with R. Each dot represents a gene. Genes displaying a variability of expression above the cut-off specified (see text) are shown in red. In the bottom of each graph the pair-wise Pearson’s correlation of all genes depicted is shown. LD, long-day; SD short-day; R1, biological replicate 1; R2, biological replicate 2.
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Figure 7: Scatter plots of gene expression expressed as log2(FPKM + 1) show more pronounced variability in long-day grown over-expressing mutant. Expression was determined from RNAseq reads for the wild-type (Columbia) and mutant (AtCHR23-4ov), with biological replicates indicated with R. Each dot represents a gene. Genes displaying a variability of expression above the cut-off specified (see text) are shown in red. In the bottom of each graph the pair-wise Pearson’s correlation of all genes depicted is shown. LD, long-day; SD short-day; R1, biological replicate 1; R2, biological replicate 2.

Mentions: Differential expression analysis using DESeq [35] or cuffdiff [36] resulted in lists of potentially differentially expressed (DE) genes. However, in additional biological replicates many of these could not be confirmed. From 96 genes identified by DESeq as potentionally DE in long-day mutant (Additional file 3), 24 genes were analyzed by qRT-PCR and 7 were confirmed as differentially expressed (33.3% of tested genes). We concluded that identified DE genes cannot be biologically validated. Further analyses therefore focused on the apparent variation in gene expression. Comparison of the expression values expressed as summed fragments per kilobase of transcript (exon model) per million mapped reads (FPKM) of replicates R1 and R2 for each sample showed the Pearson’s correlation coefficients above 0.99 (Figure 7), except for the only sample in which the growth phenotype was present: AtCHR23 over-expression in long-day conditions. In this case the data are much more disperse from the line of best fit and the Pearson’s correlation coefficient is just above 0.97 (Figure 7). In order to assess the larger between-replicate expression variability in mutant long-day, we calculated for all genes the absolute differences between the log2(FPKM + 1) expression level in the two replicates. The larger expression difference shown by the top 1% of the genes in wild-type (195 genes) was taken as cut-off for variability and used to select the number (and identity) of the genes in all other samples that showed variability higher than specified cut-off. This threshold was equivalent to an expression difference of about 1.5 fold on the normal scale. In the scatter plots of genome-wide gene expression, these genes are depicted in red (Figure 7).


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)

Scatter plots of gene expression expressed as log2(FPKM + 1) show more pronounced variability in long-day grown over-expressing mutant. Expression was determined from RNAseq reads for the wild-type (Columbia) and mutant (AtCHR23-4ov), with biological replicates indicated with R. Each dot represents a gene. Genes displaying a variability of expression above the cut-off specified (see text) are shown in red. In the bottom of each graph the pair-wise Pearson’s correlation of all genes depicted is shown. LD, long-day; SD short-day; R1, biological replicate 1; R2, biological replicate 2.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Scatter plots of gene expression expressed as log2(FPKM + 1) show more pronounced variability in long-day grown over-expressing mutant. Expression was determined from RNAseq reads for the wild-type (Columbia) and mutant (AtCHR23-4ov), with biological replicates indicated with R. Each dot represents a gene. Genes displaying a variability of expression above the cut-off specified (see text) are shown in red. In the bottom of each graph the pair-wise Pearson’s correlation of all genes depicted is shown. LD, long-day; SD short-day; R1, biological replicate 1; R2, biological replicate 2.
Mentions: Differential expression analysis using DESeq [35] or cuffdiff [36] resulted in lists of potentially differentially expressed (DE) genes. However, in additional biological replicates many of these could not be confirmed. From 96 genes identified by DESeq as potentionally DE in long-day mutant (Additional file 3), 24 genes were analyzed by qRT-PCR and 7 were confirmed as differentially expressed (33.3% of tested genes). We concluded that identified DE genes cannot be biologically validated. Further analyses therefore focused on the apparent variation in gene expression. Comparison of the expression values expressed as summed fragments per kilobase of transcript (exon model) per million mapped reads (FPKM) of replicates R1 and R2 for each sample showed the Pearson’s correlation coefficients above 0.99 (Figure 7), except for the only sample in which the growth phenotype was present: AtCHR23 over-expression in long-day conditions. In this case the data are much more disperse from the line of best fit and the Pearson’s correlation coefficient is just above 0.97 (Figure 7). In order to assess the larger between-replicate expression variability in mutant long-day, we calculated for all genes the absolute differences between the log2(FPKM + 1) expression level in the two replicates. The larger expression difference shown by the top 1% of the genes in wild-type (195 genes) was taken as cut-off for variability and used to select the number (and identity) of the genes in all other samples that showed variability higher than specified cut-off. This threshold was equivalent to an expression difference of about 1.5 fold on the normal scale. In the scatter plots of genome-wide gene expression, these genes are depicted in red (Figure 7).

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