Epigenetic marks in an adaptive water stress-responsive gene in tomato roots under normal and drought conditions

Abstract

Tolerance to water deficits was evolutionarily relevant to the conquest of land by primitive plants. In this context, epigenetic events may have played important roles in the establishment of drought stress responses. We decided to inspect epigenetic marks in the plant organ that is crucial in the sensing of drought stress: the root. Using tomato as a crop model plant, we detected the methylated epialleles of Asr2, a protein-coding gene widespread in the plant kingdom and thought to alleviate restricted water availability. We found 3 contexts (CG, CNG, and CNN) of methylated cytosines in the regulatory region of Solanum lycopersicum Asr2 but only one context (CG) in the gene body. To test the hypothesis of a link between epigenetics marks and the adaptation of plants to drought, we explored the cytosine methylation status of Asr2 in the root resulting from water-deficit stress conditions. We found that a brief exposure to simulated drought conditions caused the removal of methyl marks in the regulatory region at 77 of the 142 CNN sites. In addition, the study of histone modifications around this model gene in the roots revealed that the distal regulatory region was rich in H3K27me3 but that its abundance did not change as a consequence of stress. Additionally, under normal conditions, both the regulatory and coding regions contained the typically repressive H3K9me2 mark, which was lost after 30 min of water deprivation. As analogously conjectured for the paralogous gene Asr1, rapidly acquired new Asr2 epialleles in somatic cells due to desiccation might be stable enough and heritable through the germ line across generations, thereby efficiently contributing to constitutive, adaptive gene expression during the evolution of desiccation-tolerant populations or species.

Keywords: Asr2; epigenetics; methylation; roots; tomato; water stress.

Figure 1. Overall methylation levels in the Asr2 regulatory region and gene body. Data were grouped by methylation context in the regulatory region, which is located upstream of the transcription start site (A), and coding region (B) under both normal conditions and after 30 min of water stress. (C) Schematic representation of the entire gene, aligned to (Aand B), showing the annealing positions of the primers relative to the +1 site designed for the post-bisulfite PCR analysis. The amplicon sizes and number of cytosine residues in each context existing in the amplicons analyzed for (Aand B) are also shown. The error bars indicate the standard error (SEM). The slight decrease in the overall CNN methylation in the regulatory region was significant (*P < 0.05). The increased values in the CNG (***P < 0.001) and CNN (**P < 0.01) methylation in the coding region were also significant. The bisulfite treatments were performed as indicated in Materials and Methods. The primers for post-bisulfite PCR are listed Table S1. GenBank accession numbers for Asr2: L20756, CU468249, and X74907.

Figure 2. Detailed inspection of the stress-induced changes in asymmetric methylation (CNN) within the Asr2 regulatory region. The moving average statistical tool (http://lorien.ncl.ac.uk/ming/filter/filmav.htm) was employed. The X-axis represents 10-cytosine (5 left + 5 right) windows for each position corresponding to the 142 cytosine residues (CNN) existing in this region. Y-axis units represent the moving average of the differences between the methylation level for each cytosine under stress and the methylation level for the same cytosine under no stress. Some position numbers relative to the +1 transcription start site are indicated.

Figure 3. Histone marks on the Asr2 regulatory and coding regions. The H3K4me3 (A and B), H3K27me3 (C and D), and H3K9me2 (E and F) levels in the regulatory (A, C, and E) and coding (B, D, and F) regions under both normal conditions and after 30 min of water stress are expressed as % input. ChIP was performed as described in Materials and Methods. (G) Schematic representation of the entire gene, aligned to (A–F), showing the annealing positions of the primers designed for qPCR and the sizes of the analyzed amplicons. The error bars indicate the standard error (SEM). Statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001) histone marks are highlighted. Statistical significance between no-antibody (noise signal) and real ChiP signal is shown except for the evident H3K9me2 mark (E and F) under normal conditions (P < 0.001) for the sake of simplicity, not to mask the pronounced effect of stress on demethylation in both gene regions. The qPCR primers are listed in Table S1.

Figure 4.Asr2 expression as a consequence of water stress. The root mRNA steady-state levels were measured by qRT-PCR as described in Materials and Methods. The data were normalized to actin (A) or EF-1 (B) mRNA at each stress time before comparing the effects of the different stress treatments. Normalized against EF-1, the difference in the expression levels of Asr2 was statistically significant at 30 min (***P < 0.001). The qRT-PCR primers are listed in Table S1. The error bars indicate the standard error (SEM).