Epigenetics comprehensive experimental course based on the integration of science and education to cultivate students' ability of cutting-edge innovation.

The integration of science and education is an effective way for universities to cultivate students in cutting-edge innovative interests. Epigenetics is the expansion of classical genetics, the corresponding experimental courses of which have not been integrated into the current teaching system. In this paper, by taking advantage of our laboratory's research on the DNA methylation maintenance gene, OsMET1-2 in rice, we have integrated our innovative findings in the education curriculum, and built a comprehensive teaching system on experimentation research, which greatly stimulates the curiosity of the students. Taking the OsMET1-2 mutants and its isogenic wild-type rice plants as experimental materials, this course has successfully demonstrated a causal link between genetic mutation and epigenetic variation, a topic widely interested by the students in learning genetics and epigenetics. Through the practice of this course, students have a deeper understanding of the important role of epigenetic modifications, their scientific research capabilities have been greatly improved, thereby strongly supporting the cultivation of top innovative talents among the students.

Various potentially toxic element tolerances in different rice genotypes correlate with distinct physiological responses and alterations in DNA methylation.

Potentially toxic elements (PTEs) are harmful to plant growth and reduce crop productivity. In this work, we studied three rice genotypes (T-35, RZ-1, and RZ-2) to quantify the diverse PTE effects and tolerances by examining morphology, physiology, and DNA methylation patterns. Morphological results showed that T-35 exhibits the highest tolerance to all studied PTE stressors (Cu, Cd, Cr). Physiological responses under PTE stresses confirmed earlier findings, where T-35 showed a higher potassium (K+) content and more peroxidase (POD) accumulation in the roots than the other two rice genotypes. The differences in PTE tolerance levels observed among the three rice genotypes were also associated with variations in the heavy metal transportation (HMT) gene expression level. Moreover, methylation-sensitive blotting analysis of the selected genes showed that the DNA methylation changes occurring due to PTE treatments are mainly CHG hypomethylation in T-35 but hypermethylation in RZ-1 and RZ-2. Our results demonstrate a tight relationship among physiological response, expression levels of the HMT genes, and DNA methylation pattern under PTEs stresses. It is also indicated that plants use generic mechanisms to tolerate stresses; however, different genotypes employ different combinations of such tactics to confer tolerance, which results in diverse PTE stress tolerances. These findings shed light on the PTE stresses tolerance mechanism and help direct future breeding activities in rice.

Genome-wide locus-specific DNA methylation repatterning may facilitate rapid evolution of mercury resistance in rice.

BACKGROUND: Albeit a relatively stable epigenetic modification, DNA methylation in plants can be repatterned and play important roles in response to biotic and abiotic stresses. However, whether DNA methylation dynamics may contribute to cope with mercury (Hg) stress in plants remains to be fully investigated. OBJECTIVE: To probe the potential roles of DNA methylation dynamics in coping with Hg stress in rice. METHODS: Whole-genome bisulfite sequencing was used to profile the DNA methylation patterns of a rice Hg-resistant line (RHg) selected from a heterozygous mutant of the DNA methyltransferase 1 gene (OsMET1+/-), together with its Hg-sensitive wild-type plants of cv. Nipponbare (Nip) under both normal and Hg stress conditions. RESULTS: Genome-wide locus-specific differential methylation regions (DMRs) were detected between RHg and Nip under normal condition, the predominant DMR patterns were CG hypo-DMRs, CHG hypo-DMRs and CHH hyper-DMRs. In both lines, more hyper- than hypo-DMRs were detected at all three sequence contexts (CG, CHG and CHH) under Hg stress relative to normal condition. Comparison of DNA methylation changes in the two lines under Hg stress indicates that RHg had a more dynamic methylome than the control (Nip). Original DMRs in RHg trended to transform to opposite status (from hyper- to hypo- or vice versa) under Hg stress condition. Gene ontology analysis revealed that Hg-resistance-related DMGs were enriched in diverse biological processes. CONCLUSIONS: Our results suggest genome-wide locus-specific DNA methylation repatterning can facilitate rapid acquisition of Hg resistance in rice.

Transgenerational memory of gene expression changes induced by heavy metal stress in rice (Oryza sativa L.).

BACKGROUND: Heavy metal toxicity has become a major threat to sustainable crop production worldwide. Thus, considerable interest has been placed on deciphering the mechanisms that allow plants to combat heavy metal stress. Strategies to deal with heavy metals are largely focused on detoxification, transport and/or sequestration. The P1B subfamily of the Heavy Metal-transporting P-type ATPases (HMAs) was shown to play a crucial role in the uptake and translocation of heavy metals in plants. Here, we report the locus-specific expression changes in the rice HMA genes together with several low-copy cellular genes and transposable elements upon the heavy metal treatment and monitored the transgenerational inheritance of the altered expression states. We reveal that plants cope with heavy metal stress by making heritable changes in gene expression and further determined gene-specific responses to heavy metal stress. RESULTS: We found most HMA genes were upregulated in response to heavy metal stress, and furthermore found evidence of transgenerational memory via changes in gene regulation even after the removal of heavy metals. To explore whether DNA methylation was also altered in response to the heavy metal stress, we selected a Tos17 retrotransposon for bisulfite sequencing and studied its methylation state across three generations. We found the DNA methylation state of Tos17 was altered in response to the heavy metal stress and showed transgenerational inheritance. CONCLUSIONS: Collectively, the present study elucidates heritable changes in gene expression and DNA methylation in rice upon exposure to heavy metal stress and discusses implications of this knowledge in breeding for heavy metal tolerant crops.

Meiotic chromosome stability of a newly formed allohexaploid wheat is facilitated by selection under abiotic stress as a spandrel.

Polyploidy is a prominent route to speciation in plants; however, this entails resolving the challenges of meiotic instability facing abrupt doubling of chromosome complement. This issue remains poorly understood. We subjected progenies of a synthetic hexaploid wheat, analogous to natural common wheat, but exhibiting extensive meiotic chromosome instability, to heat or salt stress. We selected stress-tolerant cohorts and generated their progenies under normal condition. We conducted fluorescent in situ hybridization/genomic in situ hybridization-based meiotic/mitotic analysis, RNA-Seq and virus-induced gene silencing (VIGS)-mediated assay of meiosis candidate genes. We show that heritability of stress tolerance concurred with increased euploidy frequency due to enhanced meiosis stability. We identified a set of candidate meiosis genes with altered expression in the stress-tolerant plants vs control, but the expression was similar to that of common wheat (cv Chinese Spring, CS). We demonstrate VIGS-mediated downregulation of individual candidate meiosis genes in CS is sufficient to confer an unstable meiosis phenotype mimicking the synthetic wheat. Our results suggest that heritable regulatory changes of preexisting meiosis genes may be hitchhiked as a spandrel of stress tolerance, which significantly improves meiosis stability in the synthetic wheat. Our findings implicate a plausible scenario that the meiosis machinery in hexaploid wheat may have already started to evolve at its onset stage.

Mutation of the RDR1 gene caused genome-wide changes in gene expression, regional variation in small RNA clusters and localized alteration in DNA methylation in rice.

BACKGROUND: Endogenous small (sm) RNAs (primarily si- and miRNAs) are important trans/cis-acting regulators involved in diverse cellular functions. In plants, the RNA-dependent RNA polymerases (RDRs) are essential for smRNA biogenesis. It has been established that RDR2 is involved in the 24 nt siRNA-dependent RNA-directed DNA methylation (RdDM) pathway. Recent studies have suggested that RDR1 is involved in a second RdDM pathway that relies mostly on 21 nt smRNAs and functions to silence a subset of genomic loci that are usually refractory to the normal RdDM pathway in Arabidopsis. Whether and to what extent the homologs of RDR1 may have similar functions in other plants remained unknown. RESULTS: We characterized a loss-of-function mutant (Osrdr1) of the OsRDR1 gene in rice (Oryza sativa L.) derived from a retrotransposon Tos17 insertion. Microarray analysis identified 1,175 differentially expressed genes (5.2% of all expressed genes in the shoot-tip tissue of rice) between Osrdr1 and WT, of which 896 and 279 genes were up- and down-regulated, respectively, in Osrdr1. smRNA sequencing revealed regional alterations in smRNA clusters across the rice genome. Some of the regions with altered smRNA clusters were associated with changes in DNA methylation. In addition, altered expression of several miRNAs was detected in Osrdr1, and at least some of which were associated with altered expression of predicted miRNA target genes. Despite these changes, no phenotypic difference was identified in Osrdr1 relative to WT under normal condition; however, ephemeral phenotypic fluctuations occurred under some abiotic stress conditions. CONCLUSIONS: Our results showed that OsRDR1 plays a role in regulating a substantial number of endogenous genes with diverse functions in rice through smRNA-mediated pathways involving DNA methylation, and which participates in abiotic stress response.

Tissue culture-induced heritable genomic variation in rice, and their phenotypic implications.

BACKGROUND: Somaclonal variation generally occurs in plants regenerated from tissue culture. However, fundamental issues regarding molecular characteristics, mutation rates and mutation spectra of plant somatic variation as well as their phenotypic relevance have been addressed only recently. Moreover, these studies have reported highly discrepant results in different plant species and even in the same plant genotype. METHODOLOGY/PRINCIPAL FINDINGS: We investigated heritable genomic variation induced by tissue culture in rice by whole genome re-sequencing of an extensively selfed somaclonal line (TC-reg-2008) and its wild type (WT) donor (cv. Hitomebore). We computed the overall mutation rate, single nucleotide polymorphisms (SNPs), small scale insertions/deletions (Indels) and mobilization of transposable elements (TEs). We assessed chromosomal distribution of the various types of genomic variations, tested correlations between SNPs and Indels, and examined concomitancy between TE activity and its cytosine methylation states. We also performed gene ontology (GO) analysis of genes containing nonsynonymous mutations and large-effect mutations, and assayed effects of the genomic variations on phenotypes under both normal growing condition and several abiotic stresses. We found that heritable somaclonal genomic variation occurred extensively in rice. The genomic variations distributed non-randomly across each of the 12 rice chromosomes, and affected a large number of functional genes. The phenotypic penetrance of the genomic variations was condition-dependent. CONCLUSIONS/SIGNIFICANCE: Tissue culture is a potent means to generate heritable genetic variations in rice, which bear distinct difference at least in space (chromosomal distribution) from those occurred under natural settings. Our findings have provided new information regarding the mutation rate and spectrum as well as chromosomal distribution pattern of somaclonal variation in rice. Our data also suggest that rice possesses a strong capacity to canalize genetic variations under normal growing conditions to maintain phenotypic robustness, which however can be released by certain abiotic stresses to generate variable phenotypes.

Transcriptome alteration in a rice introgression line with enhanced alkali tolerance.

Alkali stress inhibits plant growth and development and thus limits crop productivity. To investigate the possible genetic basis of alkali tolerance in rice, we generated an introgressed rice line (K83) with significantly enhanced tolerance to alkali stress compared to its recipient parental cultivar (Jijing88). By using microarray analysis, we examined the global gene expression profiles of K83 and Jijing88, and found that more than 1200 genes were constitutively and differentially expressed in K83 in comparison to Jijing88 with 572 genes up- and 654 down-regulated. Upon alkali treatment, a total of 347 genes were found up- and 156 down-regulated in K83 compared to 591 and 187, respectively, in Jijing88. Among the up-regulated genes in both K83 and Jijing88, only 34 were constitutively up-regulated in K83, suggesting that both the constitutive differentially expressed genes in K83 and those induced by alkali treatment are most likely responsible for enhanced alkali tolerance. A gene ontology analysis based on all annotated, differentially expressed genes revealed that genes with expression alterations were enriched in pathways involved in metabolic processes, catalytic activity, and transport and transcription factor activities, suggesting that these pathways are associated with alkali stress tolerance in rice. Our results illuminated the novel genetic aspects of alkali tolerance in rice and established a repertory of potential target genes for biotechnological manipulations that can be used to generate alkali-tolerant rice cultivars.

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.

Changes in DNA methylation and transgenerational mobilization of a transposable element (mPing) by the topoisomerase II inhibitor, etoposide, in rice.

BACKGROUND: Etoposide (epipodophyllotoxin) is a chemical commonly used as an anti-cancer drug which inhibits DNA synthesis by blocking topoisomerase II activity. Previous studies in animal cells have demonstrated that etoposide constitutes a genotoxic stress which may induce genomic instability including mobilization of normally quiescent transposable elements (TEs). However, it remained unknown whether similar genetically mutagenic effects could be imposed by etoposide in plant cells. Also, no information is available with regard to whether the drug may cause a perturbation of epigenetic stability in any organism. RESULTS: To investigate whether etoposide could generate genetic and/or epigenetic instability in plant cells, we applied etoposide to germinating seeds of six cultivated rice (Oryza sativa L.) genotypes including both subspecies, japonica and indica. Based on the methylation-sensitive gel-blotting results, epigenetic changes in DNA methylation of three TEs (Tos17, Osr23 and Osr36) and two protein-encoding genes (Homeobox and CDPK-related genes) were detected in the etoposide-treated plants (S0 generation) in four of the six studied japonica cultivars, Nipponbare, RZ1, RZ2, and RZ35, but not in the rest japonica cultivar (Matsumae) and the indica cultivar (93-11). DNA methylation changes in the etoposide-treated S0 rice plants were validated by bisulfite sequencing at both of two analyzed loci (Tos17 and Osr36). Transpositional activity was tested for eight TEs endogenous to the rice genome in both the S0 plants and their selfed progenies (S1 and S2) of one of the cultivars, RZ1, which manifested heritable phenotypic variations. Results indicated that no transposition occurred in the etoposide-treated S0 plants for any of the TEs. Nonetheless, a MITE transposon, mPing, showed rampant mobilization in the S1 and S2 progenies descended from the drug-treated S0 plants. CONCLUSIONS: Our results demonstrate that etoposide imposes a similar genotoxic stress on plant cells as it does on animal and human cells, which may induce transgenerational genomic instability by instigating transpositional activation of otherwise dormant TEs. In addition, we show for the first time that etoposide may induce epigenetic instability in the form of altered DNA methylation patterns in eukaryotes. However, penetrance of the genotoxic effects of etoposide on plant cells, as being reflected as genetic and epigenetic instability, appears to be in a strictly genotype- and/or generation-dependent manner.

Transpositional reactivation of the Dart transposon family in rice lines derived from introgressive hybridization with Zizania latifolia.

BACKGROUND: It is widely recognized that interspecific hybridization may induce "genome shock", and lead to genetic and epigenetic instabilities in the resultant hybrids and/or backcrossed introgressants. A prominent component involved in the genome shock is reactivation of cryptic transposable elements (TEs) in the hybrid genome, which is often associated with alteration in the elements' epigenetic modifications like cytosine DNA methylation. We have previously reported that introgressants derived from hybridization between Oryza sativa (rice) and Zizania latifolia manifested substantial methylation re-patterning and rampant mobilization of two TEs, a copia retrotransposon Tos17 and a MITE mPing. It was not known however whether other types of TEs had also been transpositionally reactivated in these introgressants, their relevance to alteration in cytosine methylation, and their impact on expression of adjacent cellular genes. RESULTS: We document in this study that the Dart TE family was transpositionally reactivated followed by stabilization in all three studied introgressants (RZ1, RZ2 and RZ35) derived from introgressive hybridization between rice (cv. Matsumae) and Z. latifolia, while the TEs remained quiescent in the recipient rice genome. Transposon-display (TD) and sequencing verified the element's mobility and mapped the excisions and re-insertions to the rice chromosomes. Methylation-sensitive Southern blotting showed that the Dart TEs were heavily methylated along their entire length, and moderate alteration in cytosine methylation patterns occurred in the introgressants relative to their rice parental line. Real-time qRT-PCR quantification on the relative transcript abundance of six single-copy genes flanking the newly excised or inserted Dart-related TE copies indicated that whereas marked difference in the expression of all four genes in both tissues (leaf and root) were detected between the introgressants and their rice parental line under both normal and various stress conditions, the difference showed little association with the presence or absence of the newly mobilized Dart-related TEs. CONCLUSION: Introgressive hybridization has induced transpositional reactivation of the otherwise immobile Dart-related TEs in the parental rice line (cv. Matsumae), which was accompanied with a moderate alteration in the element's cytosine methylation. Significant difference in expression of the Dart-adjacent genes occurred between the introgressants and their rice parental line under both normal and various abiotic stress conditions, but the alteration in gene expression was not coupled with the TEs.

Spaceflight-induced genetic and epigenetic changes in the rice (Oryza sativa L.) genome are independent of each other.

An array of studies have reported that the spaceflight environment is mutagenic and may induce phenotypic and genetic changes in diverse organisms. We reported recently that in at least some plant species (e.g., rice) the spaceflight environment can be particularly potent in generating heritable epigenetic changes in the form of altered cytosine methylation patterns and activation of transposable elements. To further study the issue of spaceflight-induced genomic instability, and in particular to test whether the incurred genetic and epigenetic changes are connected or independent of each other, we performed the present study. We subjected seeds of the standard laboratory rice (Oryza sativa L.) cultivar Nipponbare to a spaceflight in the spaceship Long March 2 for 18 days. We then investigated the genetic and DNA methylation stabilities of 11 randomly selected plants germinated from the spaceflown seeds by using two kinds of DNA markers, amplified fragment length polymorphism (AFLP) and methylation sensitive amplified polymorphism (MSAP). For AFLP, by using 15 primer combinations, we assessed 460 genomic loci and found that the frequencies of genetic changes across the 11 plants ranged from 0.7% to 6.7% with an average frequency of 3.5%. For MSAP, by using 14 primer combinations, we assessed 467 loci and detected the occurrence of four major types of cytosine methylation alterations at the CCGG sites, namely CG or CNG hypomethylation and CG or CNG hypermethylation. Collectively, the frequencies of the two kinds of hypermethylation, CG (1.95%) and CNG (1.44%), are about two times higher than those of the two kinds of hypomethylation, CG (0.76%) and CNG (0.80%), though different plants showed variable frequencies for each type of alteration. Further analysis suggested that both the genetic and cytosine methylation changes manifested apparent mutational bias towards specific genomic regions, but the two kinds of instabilities are independent of each other based on correlation analysis.

The spaceflight environment can induce transpositional activation of multiple endogenous transposable elements in a genotype-dependent manner in rice.

Spaceflight represents a unique environmental condition whereby dysregulated gene expression and genomic instability can be provoked. However, detailed molecular characterization of the nature of genetic changes induced by spaceflight is yet to be documented in a higher eukaryote. Transposable elements (TEs) are ubiquitous and have played a significant role in genome evolution. Mounting evidence indicates that TEs constitute the genomic fraction that is susceptible and responsive to environmental perturbations, and hence, most likely manifesting genetic instabilities in times of stress. A predominant means for TEs to cause genetic instability is via their transpositional activation. Here we show that spaceflight has induced transposition of several endogenous TEs in rice, which belong to distinct classes including the miniature inverted terminal repeat TEs (MITEs) and long-terminal repeat (LTR) retrotransposons. Of three rice lines studied, transposition of TEs were detected in the plants germinated from space-flown dry seeds of two lines (RZ1 and RZ35), which are genetically homogeneous and stabilized recombinant inbred lines (RILs) derived from a pure-line rice cultivar, Matsumae. In contrast, the TEs remained immobile in plants derived from space-flown seeds of Matsumae itself, indicating a genotype-dependent manner of TE transposition under the spaceflight environment. Further examination showed that at least in some cases transposition of TEs was associated with cytosine demethylation within the elements. Moreover, the spaceflight-induced TE activity was heritable to organismal progenies. Thus, our results implicate that the spaceflight environment represents a potent mutagenic environment that can cause genetic instabilities by eliciting transposition of otherwise totally quiescent endogenous TEs in a higher eukaryote.

Spaceflight induces both transient and heritable alterations in DNA methylation and gene expression in rice (Oryza sativa L.).

Spaceflight represents a complex environmental condition in which several interacting factors such as cosmic radiation, microgravity and space magnetic fields are involved, which may provoke stress responses and jeopardize genome integrity. Given the inherent property of epigenetic modifications to respond to intrinsic as well as external perturbations, it is conceivable that epigenetic markers like DNA methylation may undergo alterations in response to spaceflight. We report here that extensive alteration in both DNA methylation and gene expression occurred in rice plants subjected to a spaceflight, as revealed by a set of characterized sequences including 6 transposable elements (TEs) and 11 cellular genes. We found that several features characterize the alterations: (1) All detected alterations are hypermethylation events; (2) whereas alteration in both CG and CNG methylation occurred in the TEs, only alteration in CNG methylation occurred in the cellular genes; (3) alteration in expression includes both up- and down-regulations, which did not show a general correlation with alteration in methylation; (4) altered methylation patterns in both TEs and cellular genes are heritable to progenies at variable frequencies; however, stochastic reversion to wild-type patterns and further de novo changes in progenies are also apparent; and (5) the altered expression states in both TEs and cellular genes are also heritable to selfed progenies but with markedly lower transmission frequencies than altered DNA methylation states. Furthermore, we found that a set of genes encoding for the various putative DNA methyltransferases, 5-methylcytosine DNA glycosylases, the SWI/SNF chromatin remodeller (DDM1) and siRNA-related proteins are extremely sensitive to perturbation by spaceflight, which might be an underlying cause for the altered methylation patterns in the space-flown plants. We discuss implications of spaceflight-induced epigenetic variations with regard to health safety issues of spaceship crews and potentiality of spaceflight as a means for mutagenesis in crop breeding.