Transgenerational Inheritance of Modified DNA Methylation Patterns and Enhanced Tolerance Induced by Heavy Metal Stress in Rice (<em>Oryza sativa</em> L.)

<div><h3>Background</h3><p>DNA methylation is sensitive and responsive to stressful environmental conditions. Nonetheless, the extent to which condition-induced somatic methylation modifications can impose transgenerational effects remains to be fully understood. Even less is known about the biological relevance of the induced epigenetic changes for potentially altered well-being of the organismal progenies regarding adaptation to the specific condition their progenitors experienced.</p> <h3>Methodology/Principal Findings</h3><p>We analyzed DNA methylation pattern by gel-blotting at genomic loci representing transposable elements and protein-coding genes in leaf-tissue of heavy metal-treated rice (<em>Oryza sativa</em>) plants (S0), and its three successive organismal generations. We assessed expression of putative genes involved in establishing and/or maintaining DNA methylation patterns by reverse transcription (RT)-PCR. We measured growth of the stressed plants and their unstressed progenies <em>vs.</em> the control plants. We found (1) relative to control, DNA methylation patterns were modified in leaf-tissue of the immediately treated plants, and the modifications were exclusively confined to CHG hypomethylation; (2) the CHG-demethylated states were heritable via both maternal and paternal germline, albeit often accompanying further hypomethylation; (3) altered expression of genes encoding for DNA methyltransferases, DNA glycosylase and <em>SWI/SNF</em> chromatin remodeling factor (<em>DDM1</em>) were induced by the stress; (4) progenies of the stressed plants exhibited enhanced tolerance to the same stress their progenitor experienced, and this transgenerational inheritance of the effect of condition accompanying heritability of modified methylation patterns.</p> <h3>Conclusions/Significance</h3><p>Our findings suggest that stressful environmental condition can produce transgenerational epigenetic modifications. Progenies of stressed plants may develop enhanced adaptability to the condition, and this acquired trait is inheritable and accord with transmission of the epigenetic modifications. We suggest that environmental induction of heritable modifications in DNA methylation provides a plausible molecular underpinning for the still contentious paradigm of inheritance of acquired traits originally put forward by Jean-Baptiste Lamarck more than 200 years ago.</p> </div

Alteration in DNA methylation patterns in the heavy metal-stressed plant seedlings (S0 generation) relative to the mock control (Mock) of rice ssp. <i>japonica</i>, cv. Matsumae.

<p>Gel-blotting patterns were generated by hybridizing the selected probes to DNA samples digested with a pair of methylation-sensitive isoschizomers, <i>Hpa</i>II and <i>Msp</i>I. Patterns of three of the 18 studied sequences (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041143#s4" target="_blank">Materials and methods</a>) are shown. Evidently, all alterations represent exclusive CHG hypomethylation (occurred only in <i>Msp</i>I digest). <b>A</b>, <i>Tos17</i>, which showed CHG hypomethylation in four of the eight treatments; <b>B</b>, <i>Hombox gene</i>, which showed CHG hypomethylation in five of the eight treatments; <b>C</b>, <i>CAL-11</i>, which showed CHG hypomethylation in six of the 8 treatments; <b>D</b>, <i>Hombox gene</i>, which showed complete stability in the methylation patterns in all 18 randomly chosen mock control plants (Mock S0). Arrowheads denote methylation alterations as a significant reduction in band signal intensity or complete band loss relative to the mock control.</p

Alteration in the steady-state transcript abundance of a set of nine chromatin-related genes encoding for putative DNA methyltransferases (six), 5-methylcytosine DNA glycosylase (one) and the <i>SWI/SNF</i> chromatin remodeler (<i>DDM1</i>) (two) in the heavy-metal treated rice plants at the S0, S1 and S2 generations determined by semi-quantitative RT-PCR analysis.

<p>(<b>A</b>) Transcript abundance of the nine genes in the Mock S0 and each of the four kinds of heavy-metal treatments at the indicated concentration labeled as stressed S0. (<b>B</b>) Transmission of the altered transcript abundance as well as further alteration of each of the nine genes by heavy-metal Hg²⁺ (50 µm) treatment from S0 to its eight S1 progenies. (<b>C</b>) Transmission of the altered transcript abundance of six genes in one S1 individual (S1#4) to its 14 S2 progenies. (<b>D</b>) Two examples showing lack of fluctuation for the chromatin-related genes in the untreated mock control plants across generations. For all analysis, three batches of independent RNAs were isolated from seedling-leaf tissue at the same stage, and RT-PCR was performed with each of them. The results were highly reproducible among the three independent RNA batches, and hence, only one experiment was presented. Gene name and amplification cycles are labeled. The rice <i>Actin</i> gene (Genbank accession number:X79378 ) was used as an internal control in all cases. Lack of genomic DNA was validated by the <i>Actin</i> gene on template without RT.</p

Transgenerational inheritance of the altered DNA methylation patterns induced by one (Hg²⁺) of the heavy-metal stresses at a mild concentration (50 µm) in the S0 plants (marked as S0-Hg²⁺ (50 µm) across two successive selfed generations (S1 and S2).

<p>Examples of hypomethylated patterns in the heavy-metal treated S0-generation plants, which showed “inheritance”, “new pattern” (further hypomethylation), and “reversion” to wild-type in the S1 generation (A and B), but were then stably inherited in the S2 generation (D and E). The <i>Hombox gene</i> (<b>A</b>) detected hypomethylation in S0-Hg²⁺ (50 µm) (marked with arrowhead) which in the S1 generation showed all three types of patterns, “inheritance” (plants #12 and #14, marked with arrowheads), “reversion” (plants #11 and #13, marked with arrows), and a new pattern that cannot be explained by segregation alone, and therefore most likely is associated with further methylation modification, i.e., further hypomethylation (marked with asterisks). The <i>DNA-binding protein</i><b>-</b><i>encoding gene</i> (<b>B</b>), plants #11 and #13 showed heritable DNA methylation patterns (marked with arrowhead). <b>C</b>, Hybridization of the <i>osHMA8</i> gene to genomic DNAs of 20 randomly chosen mock S1 plants which are selfed progenies of one wild-type control individual (Mock S0); monomorphic pattern indicate stable inheritance of intrinsic methylation patterns in the ground control plants. <b>D</b> and <b>E</b> are examples of changed methylation pattern in S2 generation (probe, <i>DNA-binding protein</i>). <b>D</b>, S2 generation showed complete inheritance of altered DNA methylation patterns in 20 randomly selected self-pollinated progenies of S1–4 (the upper band was lost in all plants, marked with asterisks); <b>E</b>, S2 generation showed complete inheritance of altered DNA methylation patterns in 20 randomly selected self-pollinated progenies of S1–11. <b>F</b>, Hybridization of the <i>osHMA8</i> to genomic DNAs of 20 randomly chosen Mock S2 plants which are selfed progenies of one Mock S1 individual; monomorphic patterns indicate stable inheritance of the default methylation pattern of wild-type control plants.</p

Transgenerational alteration and inheritance of DNA methylation patterns in 20 randomly chosen S1 plants derived from a Hg²⁺(50 µm)-stressed S0 individual.

<p><b>Explanation of symbols:</b> - denotes hypomethylation changes in the S0 plant or its S1 progeny plants; n denotes no methylation change in the S0 plant or its S1 progeny plants; – denotes S1 plants showing further decrease in DNA methylation; r denotes S1 plants being reverted back to the Mock control patterns; i denotes S1 plants having inherited the modified patterns of S0 plant; na denotes not applicable to the particular case.</p

Maternal vs. paternal germinal transmission of the modified DNA methylation patterns in a pair of reciprocal F1 hybrids (each crossing direction containing 20 individuals) of an unstressed wild-type control plant and a S2 individual plant (S2#7) derived from S1#4 that had originated from a Hg²⁺ (50 µm)-treated S0 plant.

<p><b>Note:</b> Compared with the Chi-square values, the <i>P</i> values of he mean frequencies greatly exceeded 0.05 indicating that there was no significant difference between maternal and paternal germinal transmission of modified DNA methylation patterns for the four studied probe.</p

Phenotypes and quantitative measurements of traits from mock control and progenies of heavy metal (Hg²⁺ at 100 µm) stressed plants under normal and heavy metal stress conditions (Hg²⁺ at 100, 300 and 500 µm, respectively).

<p>(<b>A</b>) The overall seedling phenotypes of plants (two individuals are shown) under normal (left-most) and three heavy metal (from left to right: 100, 300 and 500 µm) Hg²⁺ stress conditions, Bars = 5 cm. (<b>B</b>) Phenotypic analysis of mock control plants (Mock) and two successive selfed-pollinated progenies, S1 and S2, derived from S0-Hg²⁺ (50 µm). Seeds were germinated and seedlings grown for 10 days on Hoagland nutrient solution with or without the indicated concentrations of Hg²⁺. Plant height, fresh weight and chlorophyll content were measured. For all measurements, data are from three independent experiments each containing 10 individual plants. Error bars represent standard error (SE). * and ** denote statistical significance at 0.05 and 0.01 levels respectively in student's test.</p

Phenotypes of rice (ssp. <i>japonica</i>, cv. Matsumae) seedlings upon heavy metal stress.

<p>The inhibitory effects were manifested by growth inhibition of shoot- and/or root-length relative to that of the mock control. Type and concentration of the used heavy metals are indicated. scale bar = 5 cm.</p

Transgenerational alteration and inheritance of DNA methylation patterns in 20 randomly chosen S2 plants derived from two S1 individual plants, S1(#4) and S1(#11) which were derived from a single Hg²⁺ (50 µm)-stressed S0 individual.

<p><b>Explanation of symbols:</b> - denotes hypomethylation changes in the S0 plant or its S1 progeny plants; n denotes no methylation change in the S0 plant or its S1 progeny plants; – denotes S1 plants showing further decrease in DNA methylation; r denotes S1 plants being reverted back to the Mock control patterns; i denotes S1 plants having inherited the modified patterns of S0 plant; na denotes not applicable to the particular case.</p

DNA methylation alterations on a set of transposable elements (TEs) and protein coding genes in the somatic cells (leaves) of rice ssp. japonica cv. Matsumae germinating seedlings treated with four heavy metals (S0 plants).

<p> <b>Note:</b></p>a<p>Determined by BlastN at NCBI;</p>b<p>Changes in DNA methylation pattern is defined as: n/n: No changes in <i>Hpa</i>II and <i>Msp</i>I; n<b>/-</b>: No changes in <i>Hpa</i>II, hypomethylation in <i>Msp</i>I;</p>C<p>Among 8 kinds of treatment, the frequency which can induce the alteration on DNA methylation;</p>d<p>Among 18 sequences, the frequency of the specified treatment can induce the changes of DNA methylation.</p