Epigenetic Modifications In Plants Research Articles

The Sink-Source Relationship in Cucumber (Cucumis sativus L.) Is Modulated by DNA Methylation.

The optimization of the sink-source relationship is of great importance for crop yield regulation. Cucumber is a typical raffinose family oligosaccharide (RFO)-transporting crop. DNA methylation is a common epigenetic modification in plants, but its role in sink-source regulation has not been demonstrated in RFO-translocating species. Here, whole-genome bisulfite sequencing (WGBS-seq) was conducted to compare the nonfruiting-node leaves (NFNLs) and leaves of fruit setting (FNLs) at the 12th node by removing all female flowers in other nodes of the two treatments. We found considerable differentially methylated genes enriched in photosynthesis and carbohydrate metabolic processes. Comparative transcriptome analysis between FNLs and NFNLs indicated that many differentially expressed genes (DEGs) with differentially methylated regions were involved in auxin, ethylene and brassinolide metabolism; sucrose metabolism; and RFO synthesis pathways related to sink-source regulation. Moreover, DNA methylation levels of six sink-source-related genes in the pathways mentioned above decreased in leaves after 5-aza-dC-2'-deoxycytidine (5-Aza-dC, a DNA methyltransferase inhibitor) treatment on FNLs, and stachyose synthase (CsSTS) gene expression, enzyme activity and stachyose content in RFO synthesis pathway were upregulated, thereby increasing fruit length and dry weight. Taken together, our findings proposed an up-to-date inference for the potential role of DNA methylation in the sink-source relationship, which will provide important references for further exploring the molecular mechanism of DNA methylation in improving the yield of RFO transport plants.

Manipulating epigenetic diversity in crop plants: Techniques, challenges and opportunities

Epigenetic modifications act as conductors of inheritable alterations in gene expression, all while keeping the DNA sequence intact, thereby playing a pivotal role in shaping plant growth and development. This review article presents an overview of techniques employed to investigate and manipulate epigenetic diversity in crop plants, focusing on both naturally occurring and artificially induced epialleles. The significance of epigenetic modifications in facilitating adaptive responses is explored through the examination of how various biotic and abiotic stresses impact them. Further, environmental chemicals are explored for their role in inducing epigenetic changes, particularly focusing on inhibitors of DNA methylation like 5-AzaC and zebularine, as well as inhibitors of histone deacetylation including trichostatin A and sodium butyrate. The review delves into various approaches for generating epialleles, including tissue culture techniques, mutagenesis, and grafting, elucidating their potential to induce heritable epigenetic modifications in plants. In addition, the ground breaking CRISPR/Cas is emphasized for its accuracy in targeting specific epigenetic changes. This presents a potent tools for deciphering the intricacies of epigenetic mechanisms. Furthermore, the intricate relationship between epigenetic modifications and non-coding RNA expression, including siRNAs and miRNAs, is investigated. The emerging role of exo-RNAi in epigenetic regulation is also introduced, unveiling its promising potential for future applications. The article concludes by addressing the opportunities and challenges presented by these techniques, emphasizing their implications for crop improvement. Conclusively, this extensive review provides valuable insights into the intricate realm of epigenetic changes, illuminating their significance in phenotypic plasticity and their potential in advancing crop improvement.

Virus-Induced Gene Silencing (VIGS): A Powerful Tool for Crop Improvement and Its Advancement towards Epigenetics.

Virus-induced gene silencing (VIGS) is an RNA-mediated reverse genetics technology that has evolved into an indispensable approach for analyzing the function of genes. It downregulates endogenous genes by utilizing the posttranscriptional gene silencing (PTGS) machinery of plants to prevent systemic viral infections. Based on recent advances, VIGS can now be used as a high-throughput tool that induces heritable epigenetic modifications in plants through the viral genome by transiently knocking down targeted gene expression. As a result of the progression of DNA methylation induced by VIGS, new stable genotypes with desired traits are being developed in plants. In plants, RNA-directed DNA methylation (RdDM) is a mechanism where epigenetic modifiers are guided to target loci by small RNAs, which play a major role in the silencing of the target gene. In this review, we described the molecular mechanisms of DNA and RNA-based viral vectors and the knowledge obtained through altering the genes in the studied plants that are not usually accessible to transgenic techniques. We showed how VIGS-induced gene silencing can be used to characterize transgenerational gene function(s) and altered epigenetic marks, which can improve future plant breeding programs.

Genome-wide identification of the jumonji C domain- containing histone demethylase gene family in wheat and their expression analysis under drought stress.

Methylation and demethylation of histone play a crucial role in regulating chromatin formation and gene expression. The jumonji C (JmjC) domain-containing proteins are demethylases that are involved in regulating epigenetic modification in plants. In our study, the JmjC genes in Triticum aestivum L., Triticum turgidum L., Triticum dicoccoides L., Triticum urartu L., and Aegilops tauschii L. were identified. Phylogenetic relationship and colinearity analysis revealed that the wheat JmjC genes were conserved in A, B, and D subgenomes during evolution. Cis-acting elements analysis showed that elements related to stress response, hormone response, and light response were found in wheat JmjC genes. The expression of JmjC genes was affected by tissue types and developmental stages, and members of the same subfamily tended to have similar expression patterns in wheat. They also showed a unique expression pattern in root during PEG (Polyethylene glycol) treatment. In conclusion, comprehensive analysis indicated that three members (Tr-1A-JMJ2, Tr-1B-JMJ2, and Tr-1D-JMJ2) might be regulated by several hormones and function in the early stages of drought stress, while eight members (Tr-1B-JMJ3, Tr-4B-JMJ1, Tr-7A-JMJ1, etc.) displayed a significantly high expression after 24 h of PEG treatment, indicating a role in the later stages of drought stress. This research presents the first genome-wide study of the JmjC family in wheat, and lays the foundation for promoting the study of their functional characterization in wheat drought resistance.

Epigenetic Modifications in Plants – Impact on Phospholipid Signaling and Secondary Metabolism

Plants are considered as the "masters" of epigenetic regulation. Being exposed to many environmental factors during the vegetation period, they, have developed mechanisms on the molecular level that help them rapidly and reversibly change their epigenetic status and survive environmental stress. Recent studies document the importance of epigenetic control in plant responses to environmental stress through changes in gene activity and expression, resulting in metabolic pathways modifications as well. This brief review focuses on epigenetic modifications induced by an abiotic stress and on the effects of changes in phospholipid signalling, especially concerning the formation of secondary metabolites in plants.

Role of Epigenetics in Modulating Phenotypic Plasticity against Abiotic Stresses in Plants

Plants being sessile are always exposed to various environmental stresses, and to overcome these stresses, modifications at the epigenetic level can prove vital for their long-term survival. Epigenomics refers to the large-scale study of epigenetic marks on the genome, which include covalent modifications of histone tails (acetylation, methylation, phosphorylation, ubiquitination, and the small RNA machinery). Studies based on epigenetics have evolved over the years especially in understanding the mechanisms at transcriptional and posttranscriptional levels in plants against various environmental stimuli. Epigenomic changes in plants through induced methylation of specific genes that lead to changes in their expression can help to overcome various stress conditions. Recent studies suggested that epigenomics has a significant potential for crop improvement in plants. By the induction and modulation of various cellular processes like DNA methylation, histone modification, and biogenesis of noncoding RNAs, the plant genome can be activated which can help in achieving a quicker response against various plant stresses. Epigenetic modifications in plants allow them to adjust under varied environmental stresses by modulating their phenotypic plasticity and at the same time ensure the quality and yield of crops. The plasticity of the epigenome helps to adapt the plants during pre- and postdevelopmental processes. The variation in DNA methylation in different organisms exhibits variable phenotypic responses. The epigenetic changes also occur sequentially in the genome. Various studies indicated that environmentally stimulated epimutations produce variable responses especially in differentially methylated regions (DMR) that play a major role in the management of stress conditions in plants. Besides, it has been observed that environmental stresses cause specific changes in the epigenome that are closely associated with phenotypic modifications. However, the relationship between epigenetic modifications and phenotypic plasticity is still debatable. In this review, we will be discussing the role of various factors that allow epigenetic changes to modulate phenotypic plasticity against various abiotic stress in plants.

Redox status of the plant cell determines epigenetic modifications under abiotic stress conditions and during developmental processes

BackgroundThe oxidation-reduction (redox) status of the cell influences or regulates transcription factors and enzymes involved in epigenetic changes, such as DNA methylation, histone protein modifications, and chromatin structure and remodeling. These changes are crucial regulators of chromatin architecture, leading to differential gene expression in eukaryotes. But the cell’s redox homeostasis is difficult to sustain since the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) is not equal in plants at different developmental stages and under abiotic stress conditions. Exceeding optimum ROS and RNS levels leads to oxidative stress and thus alters the redox status of the cell. Consequently, this alteration modulates intracellular epigenetic modifications that either mitigate or mediate the plant growth and stress response. Aim of reviewRecent studies suggest that the altered redox status of the cell reform the cellular functions and epigenetic changes. Recent high-throughput techniques have also greatly advanced redox-mediated gene expression discovery, but the integrated view of the redox status, and its associations with epigenetic changes and subsequent gene expression in plants are still scarce. In this review, we accordingly focus on how the redox status of the cell affects epigenetic modifications in plants under abiotic stress conditions and during developmental processes. This is a first comprehensive review on the redox status of the cell covering the redox components and signaling, redox status alters the post-translational modification of proteins, intracellular epigenetic modifications, redox interplay during DNA methylation, redox regulation of histone acetylation and methylation, redox regulation of miRNA biogenesis, redox regulation of chromatin structure and remodeling and conclusion, future perspectives and biotechnological opportunities for the future development of the plants. Key Scientific Concepts of ReviewThe interaction of redox mediators such as ROS, RNS and antioxidants regulates redox homeostasis and redox-mediated epigenetic changes. We discuss how redox mediators modulate epigenetic changes and show the opportunities for smart use of the redox status of the cell in plant development and abiotic stress adaptation. However, how a redox mediator triggers epigenetic modification without activating other redox mediators remains yet unknown.

Epigenome editing: targeted manipulation of epigenetic modifications in plants.

Epigenetic modifications play important roles in diverse cellular processes such as X chromosome inactivation, cell differentiation, development and senescence. DNA methylation and histone modifications are major epigenetic modifications that regulate chromatin structure and gene expression without DNA sequence changes. Epigenetic alterations may induce phenotypic changes stable enough for mitotic or meiotic inheritance. Moreover, the reversibility of epigenetic marks makes the manipulation of chromatin and epigenetic signature an attractive strategy for therapeutic and breeding purposes. Targeted epigenetic manipulation, or epigenome editing, at the gene of interest commonly utilizes specific epigenetic modifiers fused with a targeting module of the conventional genome editing system. This review aims to summarize essential epigenetic components and introduce currently available epigenetic mutants and the corresponding epialleles in plants. Furthermore, advances in epigenome editing technology are discussed while proposing its potential application to plant breeding. Epimutations associated with useful traits may provide a valuable resource for crop development. It is important to explore epimutations in a variety of crop species while understanding the fundamental aspects of epigenetic regulation of agronomically important traits such as yield, quality, disease resistance and stress tolerance. In the end, plant breeding programs through epigenome editing may help not only to expand the use of limited genetic resources but also to alleviate consumers' concerns about genetically manipulated crops.

DNA methylation and histone modifications induced by abiotic stressors in plants

A review of research shows that methylation in plants is more complex and sophisticated than in microorganisms and animals. Overall, studies on the effects of abiotic stress on epigenetic modifications in plants are still scarce and limited to few species. Epigenetic regulation of plant responses to environmental stresses has not been elucidated. This study summarizes key effects of abiotic stressors on DNA methylation and histone modifications in plants. Plant DNA methylation and histone modifications in responses to abiotic stressors varied and depended on the type and level of stress, plant tissues, age, and species. A critical analysis of the literature available revealed that 44% of the epigenetic modifications induced by abiotic stressors in plants involved DNA hypomethylation, 40% DNA hypermethylation, and 16% histone modification. The epigenetic changes in plants might be underestimated since most authors used methods such as methylation-sensitive amplification polymorphism (MSAP), High performance liquid chromatography (HPLC), and immunolabeling that are less sensitive compared to bisulfite sequencing and single-base resolution methylome analyses. More over, mechanisms underlying epigenetic changes in plants have not yet been determined since most reports showed only the level or/and distribution of DNA methylation and histone modifications. Various epigenetic mechanisms are involved in response to abiotic stressors, and several of them are still unknown. Integrated analysis of the changes in the genome by omic approaches should help to identify novel components underlying mechanisms involved in DNA methylation and histone modifications associated with plant response to environmental stressors.

Understanding epigenetic modifications in response to abiotic stresses in plants

Abstract Plants have to cope up with environmental stresses during different developmental stages of their life cycle. To overcome the stresses, epigenetic regulations play a significant role in their adaptation. Epigenetic modification has already been well understood. In fact, there are numerous reports related to an epigenetic modification in plants under abiotic stresses. However, there is still a gap in understanding the correlation among different epigenetic phenomena occurring under various abiotic stresses in plants. Therefore, in this review, an attempt has been made to correlate the various epigenetic modifications in the plant under abiotic stresses. Further, recent high-throughput technologies that are being used in the epigenetic analysis have also been highlighted. Various aspects of epigenetic modification in plants have been studied giving emphasis towards abiotic stresses. DNA methylation and histone modification have been focused including the RdDM pathway, ATP-dependent chromatin remodeling, and methylation-sensitive amplified polymorphism (MSAP). With the advancement of technology, various techniques have been applied to detect DNA methylation patterns in plants. These may include bisulfite sequencing, combined bisulfite restriction analysis (COBRA), chromatin immunoprecipitation (ChIP), methylation-sensitive single nucleotide primer extension (Ms-SNuPE), cytosine assay and many more, of which bisulfite genomic sequencing, is the most commonly used. In this report, bisulfite sequencing and chromatin immunoprecipitation were described in detail. Moreover, with the advent of various bioinformatics tools and techniques, epigenetic research has been facilitated, few of which were discussed in this review.

Redox Components: Key Regulators of Epigenetic Modifications in Plants.

Epigenetic modifications including DNA methylation, histone modifications, and chromatin remodeling are crucial regulators of chromatin architecture and gene expression in plants. Their dynamics are significantly influenced by oxidants, such as reactive oxygen species (ROS) and nitric oxide (NO), and antioxidants, like pyridine nucleotides and glutathione in plants. These redox intermediates regulate the activities and expression of many enzymes involved in DNA methylation, histone methylation and acetylation, and chromatin remodeling, consequently controlling plant growth and development, and responses to diverse environmental stresses. In recent years, much progress has been made in understanding the functional mechanisms of epigenetic modifications and the roles of redox mediators in controlling gene expression in plants. However, the integrated view of the mechanisms for redox regulation of the epigenetic marks is limited. In this review, we summarize recent advances on the roles and mechanisms of redox components in regulating multiple epigenetic modifications, with a focus of the functions of ROS, NO, and multiple antioxidants in plants.

Genome-wide Profiling of Histone Lysine Butyrylation Reveals its Role in the Positive Regulation of Gene Transcription in Rice

BackgroundHistone modifications play important roles in growth and development of rice (Oryza sativa L.). Lysine butyrylation (Kbu) with a four-carbon chain is a newly-discovered histone acylation modification in rice.Main BodyIn this study, we performed chromatin immunoprecipitation sequencing (ChIP-seq) analyses, the result showed that major enrichment of histone Kbu located in genebody regions of rice genome, especially in exons. The enrichment level of Kbu histone modification is positively correlated with gene expression. Furthermore, we compared Kbu with DNase-seq and other histone modifications in rice. We found that 60.06% Kub enriched region co-located with DHSs in intergenic regions. The similar profiles were detected among Kbu and several acetylation modifications such as H3K4ac, H3K9ac, and H3K23ac, indicating that Kbu modification is an active signal of transcription. Genes with both histone Kbu and one other acetylation also had significantly increased expression compared with genes with only one acetylation. Gene Ontology (GO) enrichment analysis revealed that these genes with histone Kbu can regulate multiple metabolic process in different rice varieties.ConclusionOur study showed that the lysine butyrylation modificaiton may promote gene expression as histone acetylation and will provide resources for futher studies on histone Kbu and other epigenetic modifications in plants.

Powering Epigenetics through the 1C Pathway.

Epigenetic modifications in plants repress transposable elements to maintain genome stability and facilitate adaptation to changing environmental conditions by regulating the expression of some genes. Furthermore, conditions such as disease stress can alter the epigenetic machinery to impact gene

Methods for CpG Methylation Array Profiling Via Bisulfite Conversion.

DNA methylation is a key factor in epigenetic regulation, and contributes to the pathogenesis of many diseases, including various forms of cancers, and epigenetic events such X inactivation, cellular differentiation and proliferation, and embryonic development. The most conserved epigenetic modification in plants, animals, and fungi is 5-methylcytosine (5mC), which has been well characterized across a diverse range of species. Many technologies have been developed to measure modifications in methylation with respect to biological processes, and the most common method, long considered a gold standard for identifying regions of methylation, is bisulfite conversion. In this technique, DNA is treated with bisulfite, which converts cytosine residues to uracil, but does not affect cytosine residues that have been methylated, such as 5-methylcytosines. Following bisulfite conversion, the only cytosine residues remaining in the DNA, therefore, are those that have been methylated. Subsequent sequencing can then distinguish between unmethylated cytosines, which are displayed as thymines in the resulting amplified sequence of the sense strand, and 5-methylcytosines, which are displayed as cytosines in the resulting amplified sequence of the sense strand, at the single nucleotide level. In this chapter, we describe an array-based protocol for identifying methylated DNA regions. We discuss protocols for DNA quantification, bisulfite conversion, library preparation, and chip assembly, and present an overview of current methods for the analysis of methylation data.

Phytomelatonin: a universal abiotic stress regulator

Melatonin, a derivative of tryptophan, was first detected in plant species in 1995 and it has been shown to be a diverse regulator during plant growth and development, and in stress responses. Recently, great progress has been made towards determining the detailed functions of melatonin in plant responses to abiotic stress. Melatonin priming improves plant tolerance to cold, heat, salt, and drought stresses through regulation of genes involved in the DREB/CBF, HSF, SOS, and ABA pathways, respectively. As a scavenger of free radicals, melatonin also directly detoxifies reactive oxygen species, thus alleviating membrane oxidation. Abiotic stress-inhibited photosynthesis is partially recovered and metabolites accumulate in the presence of melatonin, leading to improved plant growth, delayed leaf senescence, and increased stress tolerance. In this review, we summarize the interactions of melatonin with phytohormones to regulate downstream gene expression, protein stabilization, and epigenetic modification in plants. Finally, we consider the need for, and approaches to, the identification of melatonin receptors and components during signaling transduction pathways.

DEMETER plant DNA demethylase induces antiviral response by interferon signalling in animal cells

DNA methylation is a prominent epigenetic modification in plants and animals regulated by similar mechanisms but the process of DNA demethylation is profoundly different. Unlike vertebrates that require a series of enzymatic conversions of 5-methylcytosine (5mC) into other bases for DNA demethylation, plants utilize the DEMETER (DME) family of 5mC DNA glycosylases to catalyze a direct removal of 5mC from DNA. Here we introduced Arabidopsis DME into human HEK-293T cells to allow direct 5mC excision, and observed that direct DNA demethylation activity was successfully implemented by DME expression. In addition, DME induced diverse cellular responses such as cell proliferation inhibition, cell cycle dysregulation and S phase arrest. Microarray and methylome analyses revealed that DME upregulated a number of genes including cell cycle components, heat shock proteins, and notably, various interferon-stimulated genes. Moreover, DME-mediated DNA demethylation activated endogenous repeat elements, which are likely to form dsRNAs as viral mimics and eventually trigger interferon cascades to establish the antiviral state. This work demonstrates that plant DNA demethylase catalyzes DNA demethylation with a bypass of initial base conversion steps, and the interferon signaling plays a pivotal role to alleviate genotoxic stresses associated with DME-induced DNA demethylation in mammalian cells.

The Influence of Polyploidy and Genome Composition on Genomic Imprinting in Mice

Genomic imprinting is an epigenetic mechanism that switches the expression of imprinted genes involved in normal embryonic growth and development in a parent-of-origin-specific manner. Changes in DNA methylation statuses from polyploidization are a well characterized epigenetic modification in plants. However, how changes in ploidy affect both imprinted gene expression and methylation status in mammals remains unclear. To address this, we used quantitative real time PCR to analyze expression levels of imprinted genes in mouse tetraploid fetuses. We used bisulfite sequencing to assess the methylation statuses of differentially methylated regions (DMRs) that regulate imprinted gene expression in triploid and tetraploid fetuses. The nine imprinted genes H19, Gtl2, Dlk1, Igf2r, Grb10, Zim1, Peg3, Ndn, and Ipw were all unregulated; in particular, the expression of Zim1 was more than 10-fold higher, and the expression of Ipw was repressed in tetraploid fetuses. The methylation statuses of four DMRs H19, intergenic (IG), Igf2r, and Snrpn in tetraploid and triploid fetuses were similar to those in diploid fetuses. We also performed allele-specific RT-PCR sequencing to determine the alleles expressing the three imprinted genes Igf2, Gtl2, and Dlk1 in tetraploid fetuses. These three imprinted genes showed monoallelic expression in a parent-of-origin-specific manner. Expression of non-imprinted genes regulating neural cell development significantly decreased in tetraploid fetuses, which might have been associated with unregulated imprinted gene expression. This study provides the first detailed analysis of genomic imprinting in tetraploid fetuses, suggesting that imprinted gene expression is disrupted, but DNA methylation statuses of DMRs are stable following changes in ploidy in mammals.

Extensive reprogramming of cytosine methylation in Oryza allotetraploids

Cytosine methylation is a prominent epigenetic modification in plants and it has been proposed to as causative for silencing some duplicates derived by polyploidization. Several synthetic polyploids have shown there is a dynamic epigenetic change following hybridization and genome doubling. Relatively few studies, however, have focused on wild natural polyploids. In this study, using a methylation-sensitive amplified fragment length polymorphism method, we investigated the levels and types of cytosine methylation in three Oryza CCDD genome allotetraploids and their related diploids with CC and EE genomes. We show an overall minor increase in methylation level in the allopolyploids, but extensive methylation reprogramming (70 %), with almost equivalently increased (22–26 %) and decreased methylation (25–27 %). Our study reveals a complicated epigenetic change in natural polyploids. The information presented in the study will be of some utility to molecular taxonomists and also rice breeders working on wide-hybridization.

Competence and regulatory interactions during regeneration in plants.

The ability to regenerate is widely exploited by multitudes of organisms ranging from unicellular bacteria to multicellular plants for their propagation and repair. But the levels of competence for regeneration vary from species to species. While variety of living cells of a plant display regeneration ability, only a few set of cells maintain their stemness in mammals. This highly pliable nature of plant cells in-terms of regeneration can be attributed to their high developmental plasticity. De novo organ initiation can be relatively easily achieved in plants by proper hormonal regulations. Elevated levels of plant hormone auxin induces the formation of proliferating mass of pluripotent cells called callus, which predominantly express lateral root meristem markers and hence is having an identity similar to lateral root primordia. Organ formation can be induced from the callus by modulating the ratio of hormones. An alternative for de novo organogenesis is by the forced expression of plant specific transcription factors. The mechanisms by which plant cells attain competence for regeneration on hormonal treatment or forced expression remain largely elusive. Recent studies have provided some insight into how the epigenetic modifications in plants affect this competence. In this review we discuss the present understanding of regenerative biology in plants and scrutinize the future prospectives of this topic. While discussing about the regeneration in the sporophyte of angiosperms which is well studied, here we outline the regenerative biology of the gametophytic phase and discuss about various strategies of regeneration that have evolved in the domain of life so that a common consensus on the entire process of regeneration can be made.

Transgenerational, Dynamic Methylation of Stomata Genes in Response to Low Relative Humidity

Transgenerational inheritance of abiotic stress-induced epigenetic modifications in plants has potential adaptive significance and might condition the offspring to improve the response to the same stress, but this is at least partly dependent on the potency, penetrance and persistence of the transmitted epigenetic marks. We examined transgenerational inheritance of low Relative Humidity-induced DNA methylation for two gene loci in the stomatal developmental pathway in Arabidopsis thaliana and the abundance of associated short-interfering RNAs (siRNAs). Heritability of low humidity-induced methylation was more predictable and penetrative at one locus (SPEECHLESS, entropy ≤ 0.02; χ2 < 0.001) than the other (FAMA, entropy ≤ 0.17; χ2 ns). Methylation at SPEECHLESS correlated positively with the continued presence of local siRNAs (r2 = 0.87; p = 0.013) which, however, could be disrupted globally in the progeny under repeated stress. Transgenerational methylation and a parental low humidity-induced stomatal phenotype were heritable, but this was reversed in the progeny under repeated treatment in a previously unsuspected manner.