Epigenetic inheritance in rice plants

Abstract

Background and aims: Epigenetics is defined as mechanisms that regulate gene expression without base sequence alteration. One molecular basis is considered to be DNA cytosine methylation, which reversibly modifies DNA or chromatin structures. Although its correlation with epigenetic inheritance over generations has been circumstantially shown, evidence at the gene level has been limited. The present study aims to find genes whose methylation status directly correlates with inheritance of phenotypic changes.

Methods: DNA methylation in vivo was artificially reduced by treating rice (Oryza sativa ssp. japonica) seeds with 5-azadeoxycytidine, and the progeny were cultivated in the field for > 10 years. Genomic regions with changed methylation status were screened by the methylation-sensitive amplified polymorphysm (MSAP) method, and cytosine methylation was directly scanned by the bisulfite mapping method. Pathogen infection with Xanthomonas oryzae pv. oryzae, race PR2 was performed by the scissors-dip method on mature leaf blades.

Key results: The majority of seedlings were lethal, but some survived to maturity. One line designated as Line-2 showed a clear marker phenotype of dwarfism, which was stably inherited by the progeny over nine generations. MSAP screening identified six fragments, among which two were further characterized by DNA blot hybridization and direct methylation mapping. One clone encoding a retrotransposon gag-pol polyprotein showed a complete erasure of 5-methylcytosines in Line-2, but neither translocation nor expression of this region was detectable. The other clone encoded an Xa21-like protein, Xa21G. In wild-type plants, all cytosines were methylated within the promoter region, whereas in Line-2, corresponding methylation was completely erased throughout generations. Expression of Xa21G was not detectable in wild type but was constitutive in Line-2. When infected with X. oryzae pv. oryzae, against which Xa21 confers resistance in a gene-for-gene manner, the progeny of Line-2 were apparently resistant while the wild type was highly susceptible without Xa21G expression.

Conclusions: These results indicated that demethylation was selective in Line-2, and that promoter demethylation abolished the constitutive silencing of Xa21G due to hypermethylation, resulting in acquisition of disease resistance. Both hypomethylation and resistant trait were stably inherited. This is a clear example of epigenetic inheritance, and supports the idea of Lamarckian inheritance which suggested acquired traits to be heritable.

Fig. 1.

Phenotypic properties. (A) Features of mature rice plants (Oryza sativa ssp. japonica, ‘Yamadanishiki’). The wild-type (WT, right) and F₇ progeny of Line-2, (Line-2, left) were cultivated under standard field conditions, and photographed at maturity. (B) Distribution of the stem length and heading days. Fifty to 130 plants each of the wild type and Line-2 were measured every year. As the representative, values for 54 F₈ Line-2 plants (Line-2) and 36 wild-type plants (WT), both harvested in 2005, are depicted for heading days (ear emergence, upper panel) and stem length (lower panel) based on the stem–leaf plot obtained from the tests for normality with 95 % confidence. The mean for the ear emergence was 101·8 ± 0·70 d for the wild type and 93·1 ± 0·98 d for Line-2. The mean for the stem length of the wild type and the Line-2 plants was 95·2 ± 4·14 and 71·4 ± 3·6 cm, respectively. (C) Relationship between the stem length and the ear emergence (heading days). The mean value for each item (ear emergence and stem length) in each year (1998–2005) was statistically calculated as described above and plotted. The Line-2 (red) samples were F₂ (1998) to F₈ (2005) (the total number was 429), and wild-type plants (green) from the corresponding years (the total number was 1017) were also measured. The horizontal and vertical axes indicate stem length and ear emergence, respectively. Note that each spot does not necessarily represent an individual plant due to many overlaps.

Fig. 2.

Methylation analysis by DNA blot hybridization. (A) Methylation status in known genes. Genomic DNA was isolated from wild type (WT) and the F₃ progeny of Line-2 plants, and a 15 µg aliquot was digested with either HpaII (H) or MspI (M). After electrophoretic fractionation on a 0·8 % agarose gel, DNA was blotted onto a nylon membrane, and subjected to hybridization with probes of known genes as indicated; OsMADS1, 25S-17S rDNA, Tos10 or Karma. (B) Methylation status in MSAP fragments. Genomic DNA from the wild type (WT) and the F₇ progeny of Line-2 plants was subjected to hybridization with DNA probes as identified; HMF1, HMF3, HMF4 or HMF6. Fragments with a changed pattern are indicated by arrows (lower panels).

Fig. 3.

Methylation analysis of the HMF2 locus. (A) Genomic organization of the retrotransposon region containing the HMF2 sequence. The number indicates the position on the BAC clone OJ1499_A07, and the restriction site position for the indicated enzymes is given on the upper panel. The positions of regions I and II, and of the hybridization probe are indicated by horizontal bars. The translated and untranslated regions are shown by filled and open blocks, respectively. (B) Inheritance of demethylation. Aliquots of 15 µg of genomic DNA isolated from wild-type (WT) plants and the indicated progeny of Line-2 (F₃–F₇) were digested with either HpaII (H) or MspI (M), and analysed by DNA blot hybridization using a unique 960 bp fragment (positions 75 340–763 00) as the probe, as shown in (A). Note that the identified fragment of 0·3 kb was the largest among CCGG-cleaved fragments which hybridized to the probe. (C) Copy number estimation and transposition. Aliquots of 15 µg of genomic DNA isolated from wild-type (WT) plants and progeny (F₄ and F₇) were digested with EcoRI, HindIII or XbaI, and analysed by DNA blot hybridization using a unique 960 bp fragment (positions 75 340–763 00) as the probe as described above. A slight difference in intensity among HindIII-cut fragments could be due to a slight difference in amounts of total DNA loaded.

Fig. 4.

Direct methylation mapping of the HMF2 region. (A) Identification of m⁵C. After bisulfite treatment, the 290 and 544 bases in regions I and II, respectively, were amplified by PCR, cloned and sequenced. Cytosines (C) and m⁵C in the original sequence were converted into thymine (T) and C, respectively. The original nucleotide sequence of the wild type without bisulfite treatment is shown on the top line with shading of the methylatable cytosines. The converted bases are indicated by T or C (shaded) in each clone originating from bisulfite-treated wild-type or dwarf progeny. Eight and 12 clones were analysed for regions I and II, respectively, for the wild-type control. For progeny analysis, ten clones for each region were prepared from each generation (F₃–F₇). The clone number is given on the left. (B) Distribution and frequency of m⁵Cs. Histograms represent the percentage of m⁵Cs over the total cytosines (vertical axis) at positions containing CpG (red), CpNpG (green) and CpNpN (blue) sequences (N is A, C or T) in the top strand at the indicated nucleotide positions (horizontal axis) in the wild type (upper panel) and Line-2 progeny (F₃–F₇) (lower panel). The bar on the top indicates the sequence shown in (A). A negative result for the progeny means no m⁵C could be assigned to these regions.

Fig. 5.

Methylation analysis of HMF5 (Xa21G). (A) Genomic organization of the Xa21 locus. A 4 kb genomic locus is illustrated with the exons (box) and introns (line) indicated. Exons (E) are numbered in Arabic letters starting from the transcription initiation site (+1 which corresponds to nucleotide position 25 208 591 of chromosome 2). CCGG sites are indicated by vertical bars over the sequence, and mapping and probed regions are indicated by horizontal bars. The regions for direct methylation mapping are located at nucleotide positions –369 to + 144 (region I), + 484 to + 808 (region II) and + 2043 to + 2413 (region III). (B) Phylogenetic relationship between Xa21G and other Xa21-related proteins. The proteins compared are D (U72726), B (Xa21) (U37133·1), C (U72723), A1 (U72725), E (U72724), A2 (U72727), F (U72728) and G (NM-001053967). (C) Inheritance of demethylation. Aliquots of 15 µg of genomic DNA isolated from the wild type (WT) and the indicated progeny (F₃–F₉) of Line-2 plants were digested with either HpaII (H) or MspI (M), and analysed by DNA blot hybridization using a unique 1 kb fragment (positions 329–1341) as the probe.

Fig. 6.

Direct methylation mapping of Xa21G. (A) Identification of m⁵C in the promoter region containing the CG island. After bisulfite treatment, the 492 bases in region I were amplified by PCR, cloned and sequenced. Cytosines (C) and m⁵C in the original sequence were converted into thymine (T) and C, respectively. The original nucleotide sequence of the wild type without bisulfite treatment is shown on the top line with shading of the methylatable cytosines. The converted bases are indicated by T or C (shaded) in each clone originating from bisulfite-treated wild type or F₉ of Line-2. Seventeen and 18 clones were analysed for the wild type and Line-2, respectively. The clone number is given on the left. (B) Distribution and frequency of m⁵Cs. Histograms represent the percentage of m⁵Cs over the total cytosines (vertical axis) at positions containing CpG (red), CpNpG (green) and CpNpN (blue) sequences (N is A, C or T) in the top strand at the indicated nucleotide positions (horizontal axis) in the wild type (upper panel) and F₉ of Line-2 (lower panel). The bar on the top indicates the sequence shown in (A). A negative result for Line-2 means that almost no m⁵C could be assigned to this region.

Fig. 7.

Expression of Xa21G. (A) Transcript accumulation profile. Total RNA was isolated from healthy leaves of wild type and Line-2 progeny (F₃–F₉), and subjected to RT–PCR with specific primers for Xa21G. As the PCR control, the actin gene was similarly amplified. (B) Response to pathogen challenge. Total RNA was isolated from healthy leaves of wild type and Line-2 (F₉), which were treated (+) or not treated (–) with Xanthomonas oryzae pv. oryzae (Xoo) for 14 d, and subjected to RT–PCR with specific primers for Xa21G and PR-1. As the control, actin cDNA and genomic DNA were amplified. (C) Resistance to X. oryzae pv. oryzae infection. Mature healthy leaf blades from the wild type (WT) or the indicated progeny of Line-2 (F₃–F₉) were inoculated with X. oryzae pv. oryzae, incubated for 16 d and photographed. Two representative samples from each plant are shown. The scale in cm is shown on the left. (D) Lesion length (cm) was measured for 3–5 independent samples 16 d after inoculation as depicted in (C) with the standard deviation. (E) Quantification of X. oryzae cells. Inoculated leaves from the indicated plants with X. oryzae were cut 20 cm from the inoculated surface after incubation for the indicated number of days, and propagated cells were counted. Values are expressed in colony-forming units (CFU) per leaf on a logarithmic scale with the standard deviation from measurements of 3–5 independent inoculations.