Hyperosmotic priming of Arabidopsis seedlings establishes a long-term somatic memory accompanied by specific changes of the epigenome

Abstract

Background: In arid and semi-arid environments, drought and soil salinity usually occur at the beginning and end of a plant's life cycle, offering a natural opportunity for the priming of young plants to enhance stress tolerance in mature plants. Chromatin marks, such as histone modifications, provide a potential molecular mechanism for priming plants to environmental stresses, but whether transient exposure of seedlings to hyperosmotic stress leads to chromatin changes that are maintained throughout vegetative growth remains unclear.

Results: We have established an effective protocol for hyperosmotic priming in the model plant Arabidopsis, which includes a transient mild salt treatment of seedlings followed by an extensive period of growth in control conditions. Primed plants are identical to non-primed plants in growth and development, yet they display reduced salt uptake and enhanced drought tolerance after a second stress exposure. ChIP-seq analysis of four histone modifications revealed that the priming treatment altered the epigenomic landscape; the changes were small but they were specific for the treated tissue, varied in number and direction depending on the modification, and preferentially targeted transcription factors. Notably, priming leads to shortening and fractionation of H3K27me3 islands. This effect fades over time, but is still apparent after a ten day growth period in control conditions. Several genes with priming-induced differences in H3K27me3 showed altered transcriptional responsiveness to the second stress treatment.

Conclusion: Experience of transient hyperosmotic stress by young plants is stored in a long-term somatic memory comprising differences of chromatin status, transcriptional responsiveness and whole plant physiology.

Figure 1

Experimental design to investigate somatic stress memory in A. thaliana. Arabidopsis thaliana plants were germinated on vertical agar plates. Growth medium supplemented with NaCl (or not, control) was applied directly to the roots (priming) of 3-week-old seedlings. After 24 h seedlings were transferred to hydroponics or to soil and grown for another 10 days without salt. A second treatment was then applied either by adding NaCl to the hydroponic solution (salt stress) or by withholding water from soil-grown plants. Epigenetic, transcriptional, and physiological differences between primed and non-primed plants were analyzed at the indicated times. Size bars in the photos: 4 cm.

Figure 2

Salt priming at seedling stage alters responses of adult plants to salt and drought. (A) Appearance of primed and non-primed (control) plants after 10 days of growth in control conditions. Plants had been subjected to a 24-h treatment with 0 (control) or 50 mM NaCl (primed) at four-leaf seedling stage on agar plates, and subsequently transferred to hydroponics. (B) Shoot and root Na content in primed (pink) and non-primed (control, blue) plants after addition of 80 mM NaCl to the hydroponic solution of plants that had grown for 10 days in control conditions after priming. Means ± SE of four individual plants are shown. Significant differences between primed and non-primed plants are indicated with * for P <0.05 and ** for P <0.01. (C) Appearance of primed and non-primed (control) plants 2 weeks after onset of drought stress. (D) Weight and seize of plants one week after onset of drought stress. Plants subjected to different concentrations of NaCl (0 to 100 mM) during the priming treatment were analyzed. Dry weight (DW) is the horizontally dashed portion of fresh weight (FW) bars (vertically dashed). Each bar is the mean of 6-10 plants ± SE. Significant differences between primed and non-primed plants are indicated with * for P <0.05 and ** for P <0.01. Asterisks apply to both FW and DW.

Figure 3

Effect of priming on genome-wide histone modification profiles. (A) Total number of continuous stretches of DNA ('islands') associated with specific histone modifications in roots of primed (PR; light colors) and non-primed (CR; dark colors) plants as determined by SICER [35]. (B) Total coverage of the genome with islands of specific histone modifications (in percent of whole genome sequence length). (C) Numbers of differences in genome-wide histone modification profiles identified by CHIPDIFF [36]. Numbers of differential sites that showed an increase of read count in the primed sample over the non-primed sample (PR/CR >1.2) are plotted to the right those that showed a decrease (CR/PR >1.2) are plotted to the left of the vertical bar. Data were obtained from pooled root material representing three independently treated plant batches of approximately 300 plants each.

Figure 4

Confirmation of individual histone methylation marks in primed roots. Position and verification of differential sites for H3K4me2 (green), H3K4me3 (red/yellow), and H3K27me3 (blue). Differences identified by CHIPDIFF are indicated with red bars above the ChIP-Seq profiles on the left (displayed in IGB). Profiles of non-primed roots (CR) are in shown in dark colors, those of primed roots (PR) in light colors. Black arrows indicate genome positions of the fragments amplified by qPCR. Average relative amounts of DNA amplified by qPCR for the indicated sites are shown in the bar graphs on the right. Each value was normalized against input and reference. References were constitutively di- or tri-methylated regions in At2g24560 (for H3K4) or At5g56920 (for H3K27). Bars are means ± SE of three independently treated replicate plant batches each consisting of approximately 300 plants (same material as pooled for ChIP-sequencing). Significant differences between primed and non-primed plants are indicated with * for P <0.05 and ** for P <0.01.

Figure 5

Effect of priming on H3K27me3 island length distribution. (A) Island length histograms plotting absolute number of H3K27m3 islands against island length in 200 bp length windows. (B) Island length histograms plotting percentage of H3K27m3 islands (relative to total island number in the sample) against island length in 200 bp windows. (C) Island length histograms for H3K4m2 and H3K4m3. (D) Island length histograms for H3K9m2. In all histograms values for non-primed root samples (CR) are given in dark colors, values for primed root samples (PR) are given in light colors.

Figure 6

Relationship between histone methylation and mRNA levels during and after priming. (A, B) Genes on the x-axis were ranked according to mRNA levels determined by RNA-Seq. The mRNA profiles shown as smooth lines were generated from plotting for each gene on the x-axis the average mRNA values (right y-axis) over the neighboring genes with ranks of +/-100. Average values of histone modification levels (A: H3K4me3, B: H3K27me3) were plotted for the same genes (left y-axis). Relationships for non-primed root samples (CR) are shown in the graphs on the left, those for primed root samples (PR) are shown in the graphs on the right. (C) Numbers of genes that show an increase (up) or decrease (down) of mRNA level (x-axis) or histone modification level (y-axis) in response to the priming treatment (primed compared to non-primed roots). Note that the majority of changes observed immediately after the priming treatment do not show the expected positive (H3K4me3) or negative (H3K27me3) correlation between mRNA and histone modification (dashed lines). (D) Short-term kinetics of changes of mRNA and H3K27me3 levels in three genes (HKT1, TEL1, and MYB75) during the priming treatment. Relative enrichment of H3K27me3 (black bars) and mRNA levels (open bars) of selected genes in roots of A. thaliana seedlings were determined by qPCR over a time course of the first 8 h (x-axis) of the priming treatment (50 mM NaCl). H3K27me3 enrichment (left y-axis) was normalized to ChIP input and to a reference region in At5g56920. mRNA levels (right y-axis) were normalized to reference gene RpII. Bars show means ± SE of four pairwise ratios of two technical replicates of qPCR carried out with pooled root material from approximately 50 plants per time point. Significant differences to time point 0 are indicated with * for P <0.01.

Figure 7

Maintenance and loss of H3K27me3 marks 10 days after priming. Average relative amounts of DNA amplified by qPCR from anti-H3K27me3 ChIP samples obtained from roots of primed (P, dark color) and non-primed (C, light colors) plants immediately after the 24-h priming treatment (24 h, blue) or 10 days later (10 d, turquoise). Each value was normalized to ChIP input and to constitutive reference region in At5g56920. Bars are means ± SE of three independently treated replicate plant batches each consisting of approximately 300 plants (same material as pooled for ChIP-sequencing). Significant differences between primed and non-primed plants are indicated with * for P <0.05 and ** for P <0.01.

Figure 8

Properties of genome-wide H3K27me3 profiles 10 days after priming. Total number of islands (A), percentage genome coverage with islands (B), island length distribution (C) and number and direction of differences between primed and non-primed samples (D) of H3K27me3 in roots of non-primed (control, dark turquoise) and primed (light turquoise) plants after a growth period of 10 days in control conditions. Data were obtained from pooled root material representing three independently treated plant batches of approximately 300 plants each. Compare to H3K27me3 immediately after the priming treatment (Figure 3A-C and Figure 5B).

Figure 9

Examples of H3K27me3 islands etching 24 h and 10 days after priming. H3K27me3 profiles of primed and non-primed (control) root samples 24 h and 10 days after priming (screenshots of IGB display). Positions and lengths of islands identified by SICER are indicated with bars in the middle section. Note that in both example regions a long H3K27me3 island in the control samples is fractionated into a shorter island in the primed samples. This effect is still apparent after a 10-day growth period in control conditions.

Figure 10

Transcript and H3K27me3 profiles of HKT1 in primed and non-primed plants. (A) mRNA levels of HKT1 (relative to constitutive gene RpII) determined by qPCR in roots of primed plants (PR, light grey and black bars) or non-primed plants (CR, white and dark grey bars) 10 days after priming and 4 h after application of 0 (-, control) or 80 mM NaCl (+, stress treatment). Inset shows very low expression of HKT1 in the shoots of the same plants. Results are shown separately for three independently primed and treated plant batches (Rep1-3) each consisting of pooled tissue from 12 plants. Bars are means ± SE of four pairwise ratios of two technical replicates. Significant differences between primed and non-primed plants for each condition (+/- salt) are indicated with * for P <0.05 or ** for P <0.01. (B) H3K27me3 profile over the HKT1 sequence in primed (PR) and non-primed (CR) root immediately after priming (24h) and 10 days later (10d) as displayed in IGB. The differential site identified by CHIPDIFF in the 24-h samples is marked with a white box.

Figure 11

Transcript profiles of PIP2E, GH3.1, and GH3.3 in primed and non-primed plants. mRNA levels of PIP2E, GH3.1, and GH3.3 (relative to constitutive gene RpII) determined by qPCR in roots of primed plants (PR, light grey and black bars) or non-primed plants (CR, white and dark grey bars) 10 days after priming and 4 h after application of 0 (-) or 80 mM NaCl (+). Bars are means ± SE of three independently treated replicate plant batches each consisting of 12 plants. Significant differences between primed and non-primed plants for each condition (+/- salt) are indicated with * for P <0.05 and (*) for P = 0.06.