Chunming Xu

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

Background 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. Methodology/Principal Findings 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 (Oryza sativa) 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 vs. 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 SWI/SNF chromatin remodeling factor (DDM1) 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. Conclusions/Significance 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.

Persistent whole-chromosome aneuploidy is generally associated with nascent allohexaploid wheat

Allopolyploidization has been a driving force in plant evolution. Formation of common wheat (Triticum aestivum L.) represents a classic example of successful speciation via allopolyploidy. Nevertheless, the immediate chromosomal consequences of allopolyploidization in wheat remain largely unexplored. We report here an in-depth investigation on transgenerational chromosomal variation in resynthesized allohexaploid wheats that are identical in genome constitution to common wheat. We deployed sequential FISH, genomic in situ hybridization (GISH), and homeolog-specific pyrosequencing, which enabled unequivocal identification of each of the 21 homologous chromosome pairs in each of >1,000 individual plants from 16 independent lines. We report that whole-chromosome aneuploidy occurred ubiquitously in early generations (from selfed generation S1 to >S20) of wheat allohexaploidy although at highly variable frequencies (20–100%). In contrast, other types of gross structural variations were scant. Aneuploidy included an unexpected hidden type, which had a euploid chromosome number of 2n = 42 but with simultaneous loss and gain of nonhomeologous chromosomes. Of the three constituent subgenomes, B showed the most lability for aneuploidy, followed by A, but the recently added D subgenome was largely stable in most of the studied lines. Chromosome loss and gain were also unequal across the 21 homologous chromosome pairs. Pedigree analysis showed no evidence for progressive karyotype stabilization even with multigenerational selection for euploidy. Profiling of two traits directly related to reproductive fitness showed that although pollen viability was generally reduced by aneuploidy, the adverse effect of aneuploidy on seed-set is dependent on both aneuploidy type and synthetic line.

Mutation of a major CG methylase in rice causes genome-wide hypomethylation, dysregulated genome expression, and seedling lethality

Significance CG cytosine methylation (mCG) is an important epigenetic marker present in most eukaryotic genomes that is maintained by an evolutionarily conserved DNA methyltransferase dubbed DNMT1 in mammals and MET1 in plants. Null mutation of DNMT1 or MET1 results in global loss of mCG and leads to embryonic death in mouse, inviability in human cancer cells, and wide-ranging developmental abnormality in Arabidopsis thaliana. This study characterizes global effects of null mutation of a MET1 gene in rice, a model plant for monocotyledons, through methylome, transcriptome, and small RNAome analyses. The findings of this study have implications for improving our understanding of the biological roles of cytosine methylation in monocots and, from an applied point of view, in epigenetic manipulation of cereal crops. Cytosine methylation at CG sites (mCG) plays critical roles in development, epigenetic inheritance, and genome stability in mammals and plants. In the dicot model plant Arabidopsis thaliana, methyltransferase 1 (MET1), a principal CG methylase, functions to maintain mCG during DNA replication, with its null mutation resulting in global hypomethylation and pleiotropic developmental defects. Null mutation of a critical CG methylase has not been characterized at a whole-genome level in other higher eukaryotes, leaving the generality of the Arabidopsis findings largely speculative. Rice is a model plant of monocots, to which many of our important crops belong. Here we have characterized a null mutant of OsMet1-2, the major CG methylase in rice. We found that seeds homozygous for OsMet1-2 gene mutation (OsMET1-2−/−), which directly segregated from normal heterozygote plants (OsMET1-2+/−), were seriously maldeveloped, and all germinated seedlings underwent swift necrotic death. Compared with wild type, genome-wide loss of mCG occurred in the mutant methylome, which was accompanied by a plethora of quantitative molecular phenotypes including dysregulated expression of diverse protein-coding genes, activation and repression of transposable elements, and altered small RNA profiles. Our results have revealed conservation but also distinct functional differences in CG methylases between rice and Arabidopsis.

Global transgenerational gene expression dynamics in two newly synthesized allohexaploid wheat (Triticum aestivum) lines

BackgroundAlteration in gene expression resulting from allopolyploidization is a prominent feature in plants, but its spectrum and extent are not fully known. Common wheat (Triticum aestivum) was formed via allohexaploidization about 10,000 years ago, and became the most important crop plant. To gain further insights into the genome-wide transcriptional dynamics associated with the onset of common wheat formation, we conducted microarray-based genome-wide gene expression analysis on two newly synthesized allohexaploid wheat lines with chromosomal stability and a genome constitution analogous to that of the present-day common wheat.ResultsMulti-color GISH (genomic in situ hybridization) was used to identify individual plants from two nascent allohexaploid wheat lines between Triticum turgidum (2n = 4x = 28; genome BBAA) and Aegilops tauschii (2n = 2x = 14; genome DD), which had a stable chromosomal constitution analogous to that of common wheat (2n = 6x = 42; genome BBAADD). Genome-wide analysis of gene expression was performed for these allohexaploid lines along with their parental plants from T. turgidum and Ae. tauschii, using the Affymetrix Gene Chip Wheat Genome-Array. Comparison with the parental plants coupled with inclusion of empirical mid-parent values (MPVs) revealed that whereas the great majority of genes showed the expected parental additivity, two major patterns of alteration in gene expression in the allohexaploid lines were identified: parental dominance expression and non-additive expression. Genes involved in each of the two altered expression patterns could be classified into three distinct groups, stochastic, heritable and persistent, based on their transgenerational heritability and inter-line conservation. Strikingly, whereas both altered patterns of gene expression showed a propensity of inheritance, identity of the involved genes was highly stochastic, consistent with the involvement of diverse Gene Ontology (GO) terms. Nonetheless, those genes showing non-additive expression exhibited a significant enrichment for vesicle-function.ConclusionsOur results show that two patterns of global alteration in gene expression are conditioned by allohexaploidization in wheat, that is, parental dominance expression and non-additive expression. Both altered patterns of gene expression but not the identity of the genes involved are likely to play functional roles in stabilization and establishment of the newly formed allohexaploid plants, and hence, relevant to speciation and evolution of T. aestivum.

Tissue-Specific Differences in Cytosine Methylation and Their Association with Differential Gene Expression in Sorghum

It has been well established that DNA cytosine methylation plays essential regulatory roles in imprinting gene expression in endosperm, and hence normal embryonic development, in the model plant Arabidopsis (Arabidopsis thaliana). Nonetheless, the developmental role of this epigenetic marker in cereal crops remains largely unexplored. Here, we report for sorghum (Sorghum bicolor) differences in relative cytosine methylation levels and patterns at 5′-CCGG sites in seven tissues (endosperm, embryo, leaf, root, young inflorescence, anther, and ovary), and characterize a set of tissue-specific differentially methylated regions (TDMRs). We found that the most enriched TDMRs in sorghum are specific for the endosperm and are generated concomitantly but imbalanced by decrease versus increase in cytosine methylation at multiple 5′-CCGG sites across the genome. This leads to more extensive demethylation in the endosperm than in other tissues, where TDMRs are mainly tissue nonspecific rather than specific to a particular tissue. Accordingly, relative to endosperm, the other six tissues showed grossly similar levels though distinct patterns of cytosine methylation, presumably as a result of a similar extent of concomitant decrease versus increase in cytosine methylation that occurred at variable genomic loci. All four tested TDMRs were validated by bisulfite genomic sequencing. Diverse sequences were found to underlie the TDMRs, including those encoding various known-function or predicted proteins, transposable elements, and those bearing homology to putative imprinted genes in maize (Zea mays). We further found that the expression pattern of at least some genic TDMRs was correlated with its tissue-specific methylation state, implicating a developmental role of DNA methylation in regulating tissue-specific or -preferential gene expression in sorghum.

Intrinsic karyotype stability and gene copy number variations may have laid the foundation for tetraploid wheat formation

Significance This paper discusses speciation by allopolyploidy of tetraploid wheat, which in turn is a milestone event in establishing hexaploid bread wheat. The results suggested that karyotype stabilization together with variation in copy number of coding genes and localized changes in genomic repeats may have contributed to the establishment of tetraploid wheat as successful species. These observations are of relevance to the plant-breeding community for developing unique wheat cultivars by wide-hybridization and chromosomal engineering. Polyploidy or whole-genome duplication is recurrent in plant evolution, yet only a small fraction of whole-genome duplications has led to successful speciation. A major challenge in the establishment of nascent polyploids is sustained karyotype instability, which compromises fitness. The three putative diploid progenitors of bread wheat, with AA, SS (S ∼ B), and DD genomes occurred sympatrically, and their cross-fertilization in different combinations may have resulted in fertile allotetraploids with various genomic constitutions. However, only SSAA or closely related genome combinations have led to the speciation of tetraploid wheats like Triticum turgidum and Triticum timopheevii. We analyzed early generations of four newly synthesized allotetraploid wheats with genome compositions SshSshAmAm, SlSlAA, SbSbDD, and AADD by combined fluorescence and genomic in situ hybridization-based karyotyping. Results of karyotype analyses showed that although SshSshAmAm and SlSlAA are characterized by immediate and persistent karyotype stability, massive aneuploidy and extensive chromosome restructuring are associated with SbSbDD and AADD in which parental subgenomes showed markedly different propensities for chromosome gain/loss and rearrangements. Although compensating aneuploidy and reciprocal translocation between homeologs prevailed, reproductive fitness was substantially compromised due to chromosome instability. Strikingly, localized genomic changes in repetitive DNA and copy-number variations in gene homologs occurred in both chromosome stable lines, SshSshAmAm and SlSlAA. Our data demonstrated that immediate and persistent karyotype stability is intrinsic to newly formed allotetraploid wheat with genome combinations analogous to natural tetraploid wheats. This property, coupled with rapid gene copy-number variations, may have laid the foundation of tetraploid wheat establishment.

Rapid genomic changes in polyploid wheat and related species: implications for genome evolution and genetic improvement

A polyploid organism by possessing more than two sets of chromosomes from one species (autopolyploidy) or two or more species (allopolyploidy) is known to have evolutionary advantages. However, by what means a polyploid accommodates increased genetic dosage or divergent genomes (allopolyploidy) in one cell nucleus and cytoplasm constitutes an enormous challenge. Recent years have witnessed efforts and progress in exploring the possible mechanisms by which these seemingly intangible hurdles of polyploidy may be ameliorated or eventually overcome. In particular, the documentation of rapid and extensive non-Mendelian genetic and epigenetic changes that often accompany nascent polyploidy is revealing: the resulting non-additive and novel gene expression at global, regional and local levels, and timely restoration of meiotic chromosomal behavior towards bivalent pairing and disomic inheritance may ensure rapid establishment and stabilization as well as its long-term evolutionary success. Further elucidation on these novel mechanisms underpinning polyploidy will promote our understanding on fundamental issues in evolutionary biology and in our manipulation capacities in future genetic improvement of important crops that are currently polyploids in genomic constitution. This review is intended to provide an updated discussion on these interesting and important issues within the scope of a specific yet one of the most important plant groups-polyploid wheat and its related species.

Genome-Wide Disruption of Gene Expression in Allopolyploids but Not Hybrids of Rice Subspecies

Hybridization and polyploidization are prominent processes in plant evolution. Hybrids and allopolyploids typically exhibit radically altered gene expression patterns relative to their parents, a phenomenon termed “transcriptomic shock.” To distinguish the effects of hybridization from polyploidization on coregulation of divergent alleles, we analyzed expression of parental copies (homoeologs) of 11,608 genes using RNA-seq-based transcriptome profiling in reciprocal hybrids and tetraploids constructed from subspecies japonica and indica of Asian rice (Oryza sativa L.). The diploid hybrids and their derived allopolyploids differ dramatically in morphology, despite having the same suite of genes and genic proportions. Allelic and homoeolog-specific transcripts were unequivocally diagnosed in the hybrids and tetraploids based on parent-specific SNPs. Compared with the in silico hybrid (parental mix), the range of progenitor expression divergence was significantly reduced in both reciprocally generated F1 hybrids, presumably due to the ameliorating effects of a common trans environment on divergent cis-factors. In contrast, parental expression differences were greatly elaborated at the polyploid level, which we propose is a consequence of stoichiometric disruptions associated with the numerous chromosomal packaging and volumetric changes accompanying nascent polyploidy. We speculate that the emergent property of “whole genome doubling” has repercussions that reverberate throughout the transcriptome and downstream, ultimately generating altered phenotypes. This perspective may yield insight into the nature of adaptation and the origin of evolutionary novelty accompanying polyploidy.

Evolution of the BBAA Component of Bread Wheat during Its History at the Allohexaploid Level

The extracted tetraploid wheat (ETW) containing the BBAA subgenomes of hexaploid bread wheat has a stabilized karyotype but anomalous phenotypes. Genome-wide comparisons between ETW and natural tetraploid wheat revealed a large number of differentially expressed genes in ETW; these changes showed early occurrence and evolutionary persistence during bread wheat evolution. Subgenome integrity in bread wheat (Triticum aestivum; BBAADD) makes possible the extraction of its BBAA component to restitute a novel plant type. The availability of such a ploidy-reversed wheat (extracted tetraploid wheat [ETW]) provides a unique opportunity to address whether and to what extent the BBAA component of bread wheat has been modified in phenotype, karyotype, and gene expression during its evolutionary history at the allohexaploid level. We report here that ETW was anomalous in multiple phenotypic traits but maintained a stable karyotype. Microarray-based transcriptome profiling identified a large number of differentially expressed genes between ETW and natural tetraploid wheat (Triticum turgidum), and the ETW-downregulated genes were enriched for distinct Gene Ontology categories. Quantitative RT-PCR analysis showed that gene expression differences between ETW and a set of diverse durum wheat (T. turgidum subsp durum) cultivars were distinct from those characterizing tetraploid cultivars per se. Pyrosequencing revealed that the expression alterations may occur to either only one or both of the B and A homoeolog transcripts in ETW. A majority of the genes showed additive expression in a resynthesized allohexaploid wheat. Analysis of a synthetic allohexaploid wheat and diverse bread wheat cultivars revealed the rapid occurrence of expression changes to the BBAA subgenomes subsequent to allohexaploidization and their evolutionary persistence.

Tissue culture-induced genetic and epigenetic alterations in rice pure-lines, F1 hybrids and polyploids

BackgroundGenetic and epigenetic alterations can be invoked by plant tissue culture, which may result in heritable changes in phenotypes, a phenomenon collectively termed somaclonal variation. Although extensive studies have been conducted on the molecular nature and spectrum of tissue culture-induced genomic alterations, the issue of whether and to what extent distinct plant genotypes, e.g., pure-lines, hybrids and polyploids, may respond differentially to the tissue culture condition remains poorly understood.ResultsWe investigated tissue culture-induced genetic and epigenetic alterations in a set of rice genotypes including two pure-lines (different subspecies), a pair of reciprocal F1 hybrids parented by the two pure-lines, and a pair of reciprocal tetraploids resulted from the hybrids. Using two molecular markers, amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP), both genetic and DNA methylation alterations were detected in calli and regenerants from all six genotypes, but genetic alteration is more prominent than epigenetic alteration. While significant genotypic difference was observed in frequencies of both types of alterations, only genetic alteration showed distinctive features among the three types of genomes, with one hybrid (N/9) being exceptionally labile. Surprisingly, difference in genetic alteration frequencies between the pair of reciprocal F1 hybrids is much greater than that between the two pure-line subspecies. Difference also exists in the pair of reciprocal tetraploids, but is to a less extent than that between the hybrids. The steady-state transcript abundance of genes involved in DNA repair and DNA methylation was significantly altered in both calli and regenerants, and some of which were correlated with the genetic and/or epigenetic alterations.ConclusionsOur results, based on molecular marker analysis of ca. 1,000 genomic loci, document that genetic alteration is the major cause of somaclonal variation in rice, which is concomitant with epigenetic alterations. Perturbed expression by tissue culture of a set of 41 genes encoding for enzymes involved in DNA repair and DNA methylation is associated with both genetic and epigenetic alterations. There exist fundamental differences among distinct genotypes, pure-lines, hybrids and tetraploids, in propensities of generating both genetic and epigenetic alterations under the tissue culture condition. Parent-of-origin has a conspicuous effect on the alteration frequencies.