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Folta et al. BMC Plant Biology 2014, 14:76 http://www.biomedcentral.com/1471-2229/14/76 RESEARCH ARTICLE Open Access Over-expression of Arabidopsis AtCHR23 chromatin remodeling ATPase results in increased variability of growth and gene expression Adam Folta1, Edouard I Severing2, Julian Krauskopf3,4, Henri van de Geest3, Jan Verver1, Jan-Peter Nap3,5 and Ludmila Mlynarova1* 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 environmentallyinduced phenotypic and transcriptional variation. Keywords: Arabidopsis, Chromatin remodeling, Growth, Gene expression, Variability, Robustness * Correspondence: ludmila.mlynarova@wur.nl 1 Laboratory of Molecular Biology, Plant Sciences Group, Wageningen University and Research Centre, Droevendaalsesteeg 1, Wageningen 6708 PB, The Netherlands Full list of author information is available at the end of the article © 2014 Folta et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Folta et al. BMC Plant Biology 2014, 14:76 http://www.biomedcentral.com/1471-2229/14/76 Background Plants have evolved finely orchestrated mechanisms to regulate their growth in response to the environment as a programmed part of their sessile life style. These mechanisms help them to cope with the (possibly adverse) environment at any period of their existence. Notably developing seedlings are vulnerable to short-term adverse environments [1,2]. As a result, plants display substantial variability of growth, a phenomenon also known as growth plasticity [3]. Such plasticity allows plants to optimize their growth and development according to the prevailing environmental conditions, ensuring the best possible strategy to complete their life cycle and propagate. Growth plasticity is potentially important for agronomic use as it affects yield and quality in unfavorable environments. Plasticity for a trait as growth is largely organized at the molecular level in which epigenetic mechanisms play a critical role [3]. Chromatin remodeling is part of the epigenetic machinery, next to DNA methylation, histone modification and small RNA-based mechanisms [4], that is an integral part of overall plant development and is associated with plant responses to biotic [5] and abiotic stress [6]. We have shown previously that the SWI/SNF2-type ATPase encoded by AtCHR12 is involved in the regulation of growth of Arabidopsis thaliana upon perceiving abiotic stress, such as drought or higher temperature [7]. Arabidopsis plants over-expressing AtCHR12 showed growth arrest of normally active primary buds, as well as reduced growth of the primary stem when stressed. Without stress, they were indistinguishable from the wild-type. The growth arrest response depended on the severity of the stress applied. Another SWI/SNF2-type ATPase, SPLAYED (SYD), was shown to be required for resistance against the necrotrophic pathogen Botrytis cinerea [5], whereas a knockout of the AtDRD1 ATPase gene showed increased susceptibility to fungal pathogen Plectosphaerella cucumerina [8]. The SWI/SNF2-type ATPases are believed to mediate the complex interplay between chromatin remodeling and the enzymes involved in DNA and histone modification. This underlines the importance of ATP-dependent chromatin remodeling in responses of plants to environmental stress. In addition, such chromatin modifications play a regulatory role during development [9] in establishing epigenetic states with expression patterns that are tightly regulated in time and space. In animals, such epigenetic states are determined early during the development, while in plants epigenetic mechanisms also operate after embryonic development [10]. Several chromatin remodeling ATPase genes have a role in plant development. The CHD3-subfamily ATPase PICKLE (PKL) selectively regulates a suite of genes during embryogenesis, seed germination and root development [11-13]. Recently, Page 2 of 18 this gene was identified as negative regulator of photomorphogenesis [14]. Out of four genes of the SWI/ SNF2-subfamily of Arabidopsis ATPases [15], SYD and BRM are involved in various, partially overlapping, developmental processes, such as root and floral development or seed maturation [16-18]. The other two members of this subfamily, AtCHR12 and AtCHR23, have roles in embryo and endosperm development. A nearly lethal atchr12/atchr23 double mutant containing weak allele displayed a variety of severe pleiotropic morphological defects, including poor maintenance of shoot and root meristems [19]. Such ATPase-mediated chromatin modification establishes a level of gene regulation that is likely to integrate developmental programs with the response to environmental signals. It is thought that epigenetic modifications help to establish a buffer against environmental perturbations [20] that results in the phenotypic robustness of the organism. Both in Drosophila [21] and in yeast [22-24] the deletion of chromatin regulator genes markedly increased the variability of the phenotype studied, indicating that proper chromatin modification may counteract genetic, environmental and/or stochastic perturbations [25,26]. We here report on the marked impact of overexpression of the AtCHR23 gene on the phenotype of Arabidopsis in terms of growth, reaction to adverse environments and genome-wide expression levels. AtCHR23 is a paralog of AtCHR12 [27] of which the effects of overexpression were presented earlier [7]. Over-expression of AtCHR23 results in reduced growth compared to wild-type Arabidopsis, but phenotypic details between AtCHR12 and AtCHR23 over-expression are notably different, showing sub-specialization of these two paralogs. The effect of AtCHR23 over-expression is notably quantitative both in terms of growth phenotype as in terms of gene expression. The over-expression of AtCHR23 increases the variability of growth and expression variability of subsets of genes in populations of identical plants. It emphasizes the important role of chromatin modification in the control of gene expression in plants. Based on these results, we propose that accurate and controlled expression of AtCHR23 is required for the stability or robustness of growth. We propose that ATCHR23-mediated chromatin remodeling could be part of a buffer system in plants that protects against environmentally-induced phenotypic and transcriptional variation [20]. Results Construction Arabidopsis mutants with altered AtCHR23 expression To generate transgenic Arabidopsis lines over-expressing the AtCHR23 gene a construct containing 35S CaMV promoter and genomic sequence of AtCHR23 (including 5’-UTR) from the accession Columbia (Additional file 1: Folta et al. BMC Plant Biology 2014, 14:76 http://www.biomedcentral.com/1471-2229/14/76 Figure S1) was used for transformation of wild-type Arabidopsis (Col-0). Two single-copy transgenic lines were identified and analyzed in detail: AtCHR23-4ov and AtCHR23-5ov. In addition, transgenic lines overexpressing cDNA copy of AtCHR23 fused in-frame to the GFP gene under the 35S CaMV promoter in front (Additional file 1: Figure S1) were generated. Two separate single-copy transgenic lines were identified and analyzed: G_AtCHR23-1ov and G_AtCHR23-3ov. A third type of over-expressing transgenic line was generated by transformation with the cDNA copy of AtCHR23 including 5’UTR fused in frame to GFP driven by the native AtCHR23-promoter (Additional file 1: Figure S1). For comparison, two loss-of-function T-DNA insertion lines affecting AtCHR23 expression were obtained from the Arabidopsis Stock Center. Both knockout lines showed no expression of full length AtCHR23 transcript. The data presented in this paper are from SALK_057856 that in the remainder of this paper will be designated as atchr23. The other insertion line gave similar results (data not shown). Over-expression of AtCHR23 reduces the growth of roots and increases phenotypic variation The growth dynamics of seedlings of the knockout (atchr23) and over-expressing lines of AtCHR23 was analyzed with the help of a root elongation assay using vertical agar plates described previously [7]. Stratified seeds of wild-type and mutant plants germinated at approximately the same time and frequency. The lengths of the primary root and hypocotyl, as well as other phenotypic characteristics, were measured repeatedly during development in different environmental conditions. To prevent possibly confounding influences of the environment experienced by the previous generation [28], all comparisons were made using seeds from parental plants (both for the wild-type and for the mutants) grown at the same time and in the same environment. Assays were based on at least 40 roots per condition, with at most 16 roots (8 mutant; 8 wild-type) per agar plate and five agar plates per assay. Clearly visible differences between different lines were observed, notably with respect to the length of the root (Figure 1A). The differences in root length depended on the environmental conditions applied. When grown at 23°C under long-day conditions, roots of the two AtCHR23-ov mutants were considerably shorter than those of Columbia wild-type (Figure 1A and B). Data is summarized in Table 1. The average length of the roots of 8-day-old wild-type seedlings was 40.7 mm, whereas of AtCHR23-4ov seedlings it was 31.9 mm (21.6% reduction) and of AtCHR23-5ov 34.6 mm (14.9% reduction). Also up-regulation of AtCHR23 with a cDNA copy of the gene (two G_AtCHR23-ov lines) resulted in seedlings with roots 14 and 22.7% shorter than wild-type, whereas Page 3 of 18 the transgenic line with the native promoter showed 11% shorter roots (Figure 1C; Table 1). In such assays, the variation in the root length was considerable, with coefficients of variation (CV) ranging from 0.161 to 0.164 for over-expressing lines, whereas for wild-type it was 0.052 (Table 1). The variation of over-expressing mutants was significantly higher than in the wild-type (Levene’s test; Table 1). These data show that upon overexpression of AtCHR23, roots become not only significantly shorter, but also more variable and less uniform. In contrast, the knockout mutant atchr23 develops roots that are only slightly longer than those of the wild-type (Figure 1B). In populations of 40 seedlings, this difference was not statistically significant. These root growth differences between the various AtCHR23 mutants and the wild-type were consistently observed in several seed stocks that were produced in various growing conditions, greenhouse or growing chambers. Moreover, similar differences and variability patterns in root length were observed in seedlings grown at 18°C and 25°C (data not shown). The variability in the phenotypic assays was assessed in more detail by analysis of the frequency distributions of the length data (Figure 2). The frequency distribution of the root lengths shows that the distribution is shifted to shorter roots when AtCHR23 is over-expressed (Figure 2A), but still quite a number of individual seedlings have roots as long as the wild-type (Figure 2A, middle two panels). Also for the distribution of the hypocotyl length, the variation is larger in populations of over-expressing seedlings than in the wild-type (Figure 2B, middle two panels). In view of all experimental efforts to standardize the environment in the phenotypic assays, we think the variation between individuals of over-expressing lines is likely to have a molecular and/or functional basis. To associate the growth arrest phenotypes with the level of AtCHR23 mRNA, the amount of AtCHR23 mRNA was determined in pools of (eight) seedlings with the help of qRT-PCR. The quantitative results are summarized in Table 1. A two-fold increase in AtCHR23 mRNA (compared to wild-type) is observed in CHR23: G_AtCHR23ov. This is apparently sufficient for the growth arrest phenotype to become detectable. Higher levels of mRNA tend to make the phenotype more pronounced, without however a clear correlation between the level of up-regulation and the length of the root. Such an association indicates a complex interplay of interactions between steady-state mRNA levels and the penetrance of the root length phenotype. The lack of correlation between root length and the level of AtCHR23 expression was also confirmed in individual seedlings of wild-type and mutant (10 seedlings of each) (data not shown). Folta et al. BMC Plant Biology 2014, 14:76 http://www.biomedcentral.com/1471-2229/14/76 Col A B Page 4 of 18 AtCHR23-4ov 50 root length (mm) 45 40 *** *** 35 30 25 20 15 10 5 0 Col C AtCHR23-4ov AtCHR23-5ov 50 45 *** *** *** 40 root length (mm) atchr23 35 30 25 20 15 10 5 3o v 23 - 1o v R 23 - H G _A tC H tC _A G C H R 23 : G _A t C R H R C ol 23 ov 0 Figure 1 Over-expression of AtCHR23 results in reduced root growth. (A) Seedlings grown for eight days at 23°C, long-day (LD). (B) Mean (± SD) length of the primary root of Columbia wild-type (Col), knockout (atchr23) and two lines over-expressing the genomic copy of AtCHR23. (C) Mean (± SD) length of the primary root of Col wild-type and lines over-expressing the cDNA copy of AtCHR23. For each line, 40 seedlings were measured. Asterisks indicate significant differences from the wild-type: ***, P < 0.001. Folta et al. BMC Plant Biology 2014, 14:76 http://www.biomedcentral.com/1471-2229/14/76 Page 5 of 18 Table 1 Root length reduction and AtCHR23 mRNA up-regulation in transgenic Arabidopsis lines with modified AtCHR23 expression Root length (mm)a CVb Columbia – WT 40.53 AtCHR23-4ov 31.89 Plant line VARc P(VAR)d 0.052 4.76 0.164 27.63 Reduction in root length (%)e Fold up-regulation AtCHR23f na na na *** 21.6 30 AtCHR23-5ov 34.65 0.161 31.32 *** 14.9 40 atchr23 41.81 0.080 12.26 * nd na G_AtCHR23-1ov 35.04 0.161 31.46 *** 14.0 15 22.7 13 11.0 2 G_AtCHR23-3ov 31.46 0.163 26.49 *** CHR23:G_AtCHR23ov 36.26 0.164 35.47 ** a Mean root length; bcoefficient of variation calculated as ratio of the standard deviation to the mean; cvariance in root length; dsignificance of variance relative to WT as determined by Levene’s test, *, P < 0.05; **, P < 0.01; ***; P < 0.001; ereduction in root length relative to WT; ffold up-regulation of AtCHR23 relative to WT. WT, wild-type; na, not applicable; nd, not detected. The reduction in root growth is due to reduced cell elongation To determine whether the reduction of root length is due to reduced cell division or reduced cell elongation, we analyzed the size of the meristematic and elongation A zone of 6-day-old seedlings. AtCHR23-4ov roots exhibited a normal cellular patterning compared to the wildtype (Figure 3A). For meristem we measured both the length of the meristematic zone and the number of meristematic cortex cells. None of them differ between B Col frequency frequency 20 15 10 5 25 30 35 40 45 10 5 5 More 7 8 9 10 More 25 frequency 20 15 10 5 20 15 10 5 0 0 25 30 35 40 45 5 More AtCHR23-5ov 6 7 8 9 10 More AtCHR23-5ov 20 25 frequency frequency 6 AtCHR23-4ov AtCHR23-4ov frequency 15 0 0 15 10 5 20 15 10 5 0 0 25 30 35 40 45 5 More 6 7 8 9 10 More 9 10 More atchr23 atchr23 20 25 frequency frequency Col 25 20 15 10 5 20 15 10 5 0 0 25 30 35 40 root length (mm) 45 More 5 6 7 8 hypocotyl length (mm) Figure 2 Frequency distribution of root (A) and hypocotyl (B) length. Seedlings (40 for each panel) were grown on agar plates for eight days at 23°C (A) or 28°C (B) in long-day conditions. In each panel, the arrow indicates the median length. Folta et al. BMC Plant Biology 2014, 14:76 http://www.biomedcentral.com/1471-2229/14/76 Page 6 of 18 Figure 3 AtCHR23 over-expression affects cell elongation. (A) Confocal images of 6-day-old Col wild-type and AtCHR23-4ov mutant roots grown at 23°C in long day conditions stained with propidium iodide. Arrows indicate the quiescent center, arrowheads indicate the boundary between the proximal meristem and elongation zone of the root. Scale bar: 50 μm. (B) Number of cells (± SD) counted in meristem (left) and mean (± SD) meristem length (right) in Col wildtype and AtCHR23-4ov mutant. (C) Mean (± SD) length of fully elongated cells in elongation zone (left) and mean (± SD) length of the elongation zone (right) in Col wild-type and AtCHR23-4ov mutant. Asterisks indicate significant differences from the wild type: ***, P < 0.001. A wild-type and mutant roots (Figure 3B). To further assess the role of cell division, we also used the cell G2-M phase cycle marker pCYCB1;1:CYCB1;1-GUS [29]. No clear difference in the pattern (Additional file1: Figure S2) and number of GUS-positive cells was observed between the wild-type and the over-expressing mutant (data not shown). This is consistent with meristem size of wild-type and mutant (Figure 3B). On the other hand, the mutant showed a significantly shortened (16.8%) elongation zone relative to the wild-type as well as reduced length (23.1%) of the fully elongated cells (Figure 3C). Taken together, these results indicate that the major effect of AtCHR23 up-regulation in the root is the reduction of cell elongation. Col B AtCHR23-4ov Meristem size 50 45 Col AtCHR23-4ov Meristem length (µm) 35 30 25 20 15 10 5 0 250 200 150 100 50 0 Elongation zone length 180 160 Col AtCHR23-4ov 140 120 100 80 60 40 20 0 *** 1600 1400 Elongation zone length (µm) C Cell length (µm) Col AtCHR23-4ov 300 40 Number of cells 350 1200 1000 800 600 400 200 0 Col AtCHR23-4ov *** Over-expression of AtCHR23 results in smaller seedlings and smaller plantlets Analyses of two AtCHR23-ov lines demonstrate that over-expression of AtCHR23 also resulted in overall reduced seedling and plant growth (Figure 4). Overexpressing lines showed reduced growth of the cotyledon (Figure 4A) and hypocotyl (Figure 4B). The mean cotyledon area was reduced from 4.67 mm2 in the wildtype to 3.35 mm2 in AtCHR23-4ov (28.3% reduction) and to 3.83 mm2 in AtCHR23-5ov (18% reduction). The length of the hypocotyls was determined from seedlings grown at 25°C or 28°C. The latter temperature is known to induce considerable hypocotyl elongation [30]. The average hypocotyl length of 25°C-grown 8-day-old seedlings of over-expressing lines was reduced to 1.97 mm (about 20% reduction) compared to 2.42 mm of the wild-type, while the length of the hypocotyl of the knockout did not differ significantly from the wild-type. Such differences become more obvious at 28°C (Figure 4B). Both temperatures show that up-regulation of AtCHR23 leads to a significant overall reduction in the growth of seedlings. The increased growth variability of mutants cotyledon and hypocotyl was not significant (Levene’s test; Additional file 2: Table S1). To determine if and how the effects on plant size due to AtCHR23 over-expression generate phenotypic changes further in development, two parameters for vegetative growth were measured in soil-grown plants: the leaf area Folta et al. BMC Plant Biology 2014, 14:76 http://www.biomedcentral.com/1471-2229/14/76 Page 7 of 18 B 7 12 6 10 5 *** hypocotyl length (mm) cotyledon area (mm2) A *** 4 3 2 1 0 Col AtCHR23-4ov AtCHR23-5ov atchr23 6 4 *** *** 2 0 Col AtCHR23-4ov AtCHR23-5ov atchr23 C 25ºC 28ºC D 45 20 18 40 ** rosette diameter (mm) ** 16 14 leaf area (mm2) *** *** 8 12 10 8 6 *** 30 25 20 15 4 10 2 5 0 * 35 0 Col AtCHR23-4ov AtCHR23-5ov atchr23 Col AtCHR23-4ov AtCHR23-5ov atchr23 Figure 4 Over-expression of AtCHR23 leads to overall reduced seedling and plant growth. (A) Mean (± SD) cotyledon area of 8-day-old wild-type (Col) and mutant seedlings grown at 25°C in long day conditions. (B) Mean (± SD) of hypocotyl length of wild-type (Col) and mutant plants grown for 8 days at 25°C or 28°C in long-day conditions. (C) Mean (± SD) leaf area of first rosette leaf of 15-day-old soil grown wild-type (Col) and mutant plants in long- day conditions. (D) Mean (± SD) rosette diameter of 4-week-old wild-type (Col) and mutant plants grown as in (C). For each line, 40 seedlings or 15 plants were measured. Asterisks indicate significant differences from the wild type: **, P < 0.01; ***, P < 0.001. and the diameter of the rosette. Both parameters were determined from digital images of 15 soil-grown plants. The average surface area of the first rosette leaf of the wildtype was 15.7 mm2. This was reduced to 13.5 mm2 in AtCHR23-4ov and to 14.0 mm2 in AtCHR23-5ov, so overexpressing lines have up to 15% smaller leaves than the wild-type (Figure 4C). The knockout line had slightly larger leaves (5%), but again this difference was not statistically significant in the experimental set-up chosen. Similar growth differences were observed for the third rosette leaf (data not shown). Leaves of over-expressing mutants also showed significantly increased growth variability relative to wild-type (Levene’s test; Additional file 2: Table S1). Furthermore, the average rosette diameter of 4-week-old over-expressing mutants was reduced in size (Figure 4D). While the wild-type rosette diameter was 34.1 mm, it was 27.2 mm in AtCHR23-4ov and 30.1 mm in AtCHR23-5ov. Compared to the wild-type it represents 20% and 12% reduction in the size of the rosette in the mutants, respectively. It shows that also during vegetative development plants over-expressing AtCHR23 tend to stay smaller than the wild-type. Light conditions determine the growth characteristics of over-expressing lines As light is a crucial environmental factor affecting plant growth [31], we evaluated the growth dynamics of the various AtCHR23 expression variants under different light regimes. In continuous light, all AtCHR23 mutants confirm the pattern of root length as presented above for long-day conditions. Over-expressing lines have a significantly reduced root length relative to the wildtype, whereas the knockout tends to have (in this case indeed significantly) longer roots (Figure 5A). In the dark, however, none of the lines significantly differed in root length from that of wild-type (Figure 5B). In the dark, root growth is known to be significantly reduced [32,33], while the hypocotyl is known to elongate (etiolate) more than in the light [34]. Establishing further reductions in root length in such an environment is therefore less reliable. However, also the length of the hypocotyl of seedlings grown in the dark at either 23°C or 28°C (Figure 5B) was not different from the wild-type. Also at short day conditions (10 days at 8 h light/16 h dark at 23°C; Figure 5C), the length of neither roots nor hypocotyls of mutants could be distinguished from the wild-type. One possible cause for the lack of the phenotype in dark and short-day could be the instabilities of AtCHR23 mRNA over-expression. However, quantitative expression analysis of AtCHR23 in dark and short-day grown seedlings confirmed the same level of upregulation relative to wild-type as in long-day (data not shown). The lack of phenotype in dark and short-day grown mutants cannot be therefore explained by reduced Folta et al. BMC Plant Biology 2014, 14:76 http://www.biomedcentral.com/1471-2229/14/76 A Page 8 of 18 Abiotic stress magnifies the impact of AtCHR23 over-expression 50 Continuous light 45 *** 40 *** *** root length (mm) 35 30 25 20 15 10 5 0 Col AtCHR23-4ov AtCHR23-5ov atchr23 B 25 Dark Series1 Col Series2 AtCHR23-4ov Series3 AtCHR23-5ov Series4 atchr23 length (mm) 20 15 10 5 0 hypocotyl-23°C hypocotyl-28°C root-23°C C 50 45 Short-day Col Series1 AtCHR23-4ov Series2 AtCHR23-5ov Series3 atchr23 Series4 40 length (mm) 35 30 25 20 15 10 5 0 root-23°C hypocotyl-23°C Figure 5 AtCHR23 over-expression only affects root length in sufficient light. (A) Mean (± SD) root length of wild-type (Col) and mutant seedlings grown for 10 days at 23°C in continuous light. (B) Mean (± SD) root and hypocotyl length of 10-day-old wild-type (Col) and mutant seedlings grown at the indicated temperature in the dark. (C) Mean (± SD) root and hypocotyl length of 10-day-old wildtype (Col) and mutant seedlings grown at 23°C in short-day conditions. For each line 40 seedlings were measured. Asterisks indicate significant differences from the wild type: ***, P < 0.001. levels of AtCHR23 over-expression. These results show that light markedly influences the impact of modified AtCHR23 expression on the growth dynamics of Arabidopsis seedlings: sufficient (amounts of) light is required to establish the AtCHR23-mediated growth phenotype. 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 AtCHR234ov 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 overexpression on the seedlings growth and that the effect extends beyond the seedling stage. Genome-wide RNAseq analysis demonstrates increased variability of gene expression upon AtCHR23 over-expression The growth phenotype conferred by AtCHR23 overexpression was evaluated by RNA sequencing. Two biological replicates of pooled eight-day-old seedlings of Folta et al. BMC Plant Biology 2014, 14:76 http://www.biomedcentral.com/1471-2229/14/76 A Page 9 of 18 B NaCl Col AtCHR23-4ov 40 NaCl root length (mm) 35 30 *** *** 25 20 15 10 5 0 Col C Mannitol Col AtCHR23-4ov D AtCHR23-4ov AtCHR23-5ov 40 atchr23 Mannitol ** root length (mm) 35 30 *** *** 25 20 15 10 5 0 Col E NaCl Col AtCHR23-4ov F AtCHR23-4ov AtCHR23-5ov 40 NaCl 35 rosette diameter (mm2) atchr23 *** 30 25 20 15 10 5 0 Col AtCHR23-4ov 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-weekold 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. AtCHR23-4ov and the wild-type (Columbia) grown at 23°C in long-day (with the reduced growth phenotype) and short-day (without the reduced growth phenotype) photoperiods were evaluated. For each of the eight samples, more than 60 million reads were generated. Given the experimental set-up, expression differences associated with the reduced growth phenotype were expected between the over-expressing line in long-day conditions relative to all other samples. 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 Folta et al. BMC Plant Biology 2014, 14:76 http://www.biomedcentral.com/1471-2229/14/76 Page 10 of 18 AtCHR23-4ov LD 10 12 14 8 6 r = 0.971 0 0 r = 0.995 4 6 8 10 12 14 0 2 4 6 8 10 log2(FPKM + 1)_R1 Columbia SD AtCHR23-4ov SD 8 6 2 log2(FPKM + 1)_R2 8 6 4 2 r = 0.993 0 0 r = 0.994 14 10 12 14 log2(FPKM + 1)_R1 12 4 2 10 12 14 0 log2(FPKM + 1)_R2 4 2 log2(FPKM + 1)_R2 10 12 14 8 6 4 2 log2(FPKM + 1)_R2 Columbia LD 0 2 4 6 8 10 12 14 log2(FPKM + 1)_R1 0 2 4 6 8 10 12 14 log2(FPKM + 1)_R1 Figure 7 Scatter plots of gene expression expressed as log2(FPKM + 1) show more pronounced variability in long-day grown overexpressing 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. 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). In long-day conditions, the AtCHR23 over-expressing mutant has no less than 2007 genes with larger variation (Figure 8A). Of these, 68 genes were also variable in wild-type (Figure 8; Additional file 4). This shows that AtCHR23 over-expression increases the expression variability of a considerable subgroup of genes compared to the wild-type. In contrast, in short-day conditions, 381 genes were identified as variable in the wild-type, whereas 276 genes were identified in the mutant line, of which 82 were shared (Figure 8B; Additional file 4). The larger subgroup of variable genes is therefore associated with the higher over-expression of AtCHR23 observed in long-day conditions. This may point to a causal relationship between AtCHR23 over-expression and increased variability of gene expression. The 68 long-day variable genes shared between the wild-type and the mutant are less correlated between the two replicates of AtCHR23 over-expressing mutant (R2 = 0.038) relative to the wildtype (R2 = 0.625) (Figure 9). It indicates that the expression of genes which are already noisy in natural conditions (the wild-type) become even more noisy when AtCHR23 is over-expressed. To evaluate the function of the genes with higher variation in gene expression when AtCHR23 is overexpressed, gene ontology (GO) analysis was performed. For this, the subset of 298 genes (from the 2007) was selected that had at least 3-fold expression difference between the two biological replicates. Genes were classified using the Classification SuperViewer [37] as being overor under-represented. The main results are summarized in Additional file 1: Figure S4. Biological Process subcategories that were over-represented include responses to stress, stress stimuli and developmental processes, in