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.

Background: mPingis an endogenous MITE in the rice genome, which is quiescent under normal conditions but can be induced towards mobilization under various stresses. The cellular mechanism responsible for modulating the activity ofmPingremains unknown. Cytosine methylation is a major epigenetic modification in most eukaryotes, and the primary function of which is to serve as a genome defense system including taming activity of transposable elements (TEs). Given that tissue-culture is capable of inducing both methylation alteration andmPingtransposition in certain rice genotypes, it provides a tractable system to investigate the possible relationship between the two phenomena.Results: mPingtransposition and cytosine methylation alteration were measured in callus and regenerated plants in three rice (ssp.indica) genotypes, V14, V27 and R09. All three genotypes showed transposition ofmPing, though at various frequencies. Cytosine methylation alteration occurred both at themPing-flanks and at random loci sampled globally in callus and regenerated plants of all three genotypes. However, a sharp difference in the changing patterns was noted between themPing-flanks and random genomic loci, with a particular type of methylation modification, i.e., CNG hypermethylation, occurred predominantly at themPing-flanks. Pearson's test on pairwise correlations indicated thatmPingactivity is positively correlated with specific patterns of methylation alteration at random genomic loci, while the element's immobility is positively correlated with methylation levels of themPing's 5'-flanks. Bisulfite sequencing of twomPing-containing loci showed that whereas for the immobile locus loss of CG methylation in the 5'-flank was accompanied by an increase in CHG methylation, together with an overall increase in methylation of all three types (CG, CHG and CHH) in themPing-body region, for the active locus erasure of CG methylation in the 5'-flank was not followed by such a change.Conclusion: Our results documented that tissue culture-inducedmPingactivity in rice ssp.indicais correlated with alteration in cytosine methylation patterns at both random genomic loci and the elements' flanks, while the stability ofmPingpositively correlates with enhanced methylation levels of both the flanks and probably the elementsper se. Thus, our results implicate a possible role of cytosine methylation in maintainingmPingstability under normal conditions, and in releasing the element's activity as a consequence of epigenetic perturbation in a locus-specific manner under certain stress conditions.

Background: Inter-specific hybridization occurs frequently in plants, which may induce genetic and epigenetic instabilities in the resultant hybrids, allopolyploids and introgressants. It remains unclear however whether pollination by alien pollens of an incompatible species may impose a "biological stress" even in the absence of genome-merger or genetic introgression, whereby genetic and/or epigenetic instability of the maternal recipient genome might be provoked. Results: We report here the identification of a rice mutator-phenotype from a set of rice plants derived from a crossing experiment involving two remote and apparently incompatible species, Oryza sativa L. and Oenothera biennis L. The mutator-phenotype (named Tong211-LP) showed distinct alteration in several traits, with the most striking being substantially enlarged panicles. Expectably, gel-blotting by total genomic DNA of the pollen-donor showed no evidence for introgression. Characterization of Tong211-LP (S0) and its selfed progenies (S1) ruled out contamination (via seed or pollen) or polyploidy as a cause for its dramatic phenotypic changes, but revealed transgenerational mobilization of several previously characterized transposable elements (TEs), including a MITE (mPing), and three LTR retrotransposons (Osr7, Osr23 and Tos17). AFLP and MSAP fingerprinting revealed extensive, transgenerational alterations in cytosine methylation and to a less extent also genetic variation in Tong211-LP and its immediate progenies. mPing mobility was found to correlate with cytosine methylation alteration detected by MSAP but not with genetic variation detected by AFLP. Assay by q-RT-PCR of the steady-state transcript abundance of a set of genes encoding for the various putative DNA methyltransferases, 5-methylcytosine DNA glycosylases, and small interference RNA (siRNA) pathway-related proteins showed that, relative to the rice parental line, heritable perturbation in expression of 12 out of the 13 genes occurred in the mutator-phenotype and its sefled progenies. Conclusion: Transgenerational epigenetic instability in the form of altered cytosine methylation and its associated TE activity occurred in a rice mutator-phenotype produced by pollinating the rice stigma with pollens of O. biennis. Heritably perturbed homeostatic expression-state of genes involved in maintenance of chromatin structure is likely an underlying cause for the alien pollination-induced transgenerational epigenetic/genetic instability, and which occurred apparently without entailing genome merger or genetic introgression.

Elevated near-surface ozone (O₃) concentrations have surpassed the tolerance limits of plants, significantly impacting crop growth and yield. To mitigate ozone pollution, plants must evolve a rapid and effective defense mechanism to alleviate ozone-induced damage. DNA methylation, as one of the most crucial epigenetic modifications, plays a pivotal role in maintaining gene stability, regulating gene expression, and enhancing plant resilience to environmental stressors. However, the epigenetic response of plants to O₃ stress, particularly DNA methylation variations and their intergenerational transmission, remains poorly understood. This study aims to explore the epigenetic mechanisms underlying plant responses to ozone stress across generations and to identify potential epigenetic modification sites or genes crucial in response to ozone stress. Using Open Top Chambers (OTCs), we simulated ozone conditions and subjected foxtail millet to continuous ozone stress at 200 nmol mol^-1 for two consecutive generations (S0 and S1). Results revealed that under high-concentration ozone stress, foxtail millet leaves exhibited symptoms ranging from yellowing and curling to desiccation, but the damage in the S1 generation was not more severe than that in the S0 generation. Methylation Sensitive Amplified Polymorphism (MSAP) analysis of the two generations indicated that ozone stress-induced methylation variations ranging from 10.82% to 13.59%, with demethylation events ranged from 0.52% to 5.58%, while hypermethylation occurred between 0.35% and 2.76%. Reproductive growth stages were more sensitive to ozone than vegetative stages. Notably, the S1 generation exhibited widespread demethylation variations, primarily at CNG sites, compared to S0 under similar stress conditions. The inheritance pattern between S0 and S1 generations was mainly of the A-A-B-A type. By recovering and sequencing methylation variant bands, we identified six stress-related differential amplification sequences, implicating these variants in various biological processes. These findings underscore the potential significance of DNA methylation variations as a critical mechanism in plants’ response to ozone stress, providing theoretical insights and references for a comprehensive understanding of plant adaptation mechanisms to ozone stress and the epigenetic role of DNA methylation in abiotic stress regulation.

Organelle-derived nuclear DNAs, nuclear plastid DNAs (NUPTs), and nuclear mitochondrial DNAs (NUMTs) have been identified in plants. Most, if not all, genes residing in NUPTs/NUMTs (NUPGs/NUMGs) are known to be inactivated and pseudogenized. However, the role of epigenetic control in silencing NUPGs/NUMGs and the dynamic evolution of NUPTs/NUMTs with respect to organismal phylogeny remain barely explored. Based on the available nuclear and organellar genomic resources of wheat (genus Triticum) and goat grass (genus Aegilops) within Triticum/Aegilops complex species, we investigated the evolutionary fates of NUPTs/NUMTs in terms of their epigenetic silencing and their dynamic occurrence rates in the nuclear diploid genomes and allopolyploid subgenomes. NUPTs and NUMTs possessed similar genomic atlas, including (i) predominantly located in intergenic regions and preferential integration to gene regulation regions and (ii) generating sequence variations in the nuclear genome. Unlike nuclear indigenous genes, the alien NUPGs/NUMGs were associated with repressive epigenetic signals, namely high levels of DNA methylation and low levels of active histone modifications. Phylogenomic analyses suggested that the species-specific and gradual accumulation of NUPTs/NUMTs accompanied the speciation processes. Moreover, based on further pan-genomic analyses, we found significant subgenomic asymmetry in the NUPT/NUMT occurrence, which accumulated during allopolyploid wheat evolution. Our findings provide insight into the dynamic evolutionary fates of organelle-derived nuclear DNA in plants.

The NDPK gene family is an important group of genes in plants, playing a crucial role in regulating energy metabolism, growth, and differentiation, cell signal transduction, and response to abiotic stress. However, our understanding of the NDPK gene family in Brassica napus L. remains limited. This paper systematically analyzes the NDPK gene family in B. napus, particularly focusing on the evolutionary differences within the species. In this study, sixteen, nine, and eight NDPK genes were identified in B. napus and its diploid ancestors, respectively. These genes are not only homologous but also highly similar in their chromosomal locations. Phylogenetic analysis showed that the identified NDPK proteins were divided into four clades, each containing unique motif sequences, with most NDPKs experiencing a loss of introns/exons during evolution. Collinearity analysis revealed that the NDPK genes underwent whole-genome duplication (WGD) events, resulting in duplicate copies, and most of these duplicate genes were subjected to purifying selection. Cis-acting element analysis identified in the promoters of most NDPK genes elements related to a light response, methyl jasmonate response, and abscisic acid response, especially with an increased number of abscisic acid response elements in B. napus. RNA-Seq results indicated that NDPK genes in B. napus exhibited different expression patterns across various tissues. Further analysis through qRT-PCR revealed that BnNDPK genes responded significantly to stress conditions such as salt, drought, and methyl jasmonate. This study enhances our understanding of the NDPK gene family in B. napus, providing a preliminary theoretical basis for the functional study of NDPK genes and offering some references for further revealing the phenomenon of polyploidization in plants.