Frequency of cytosine methylation in the adjacent regions of soybean retrotransposon SORE-1 depends on chromosomal location.

Mobilization of transposable elements (TEs) is suppressed by epigenetic mechanisms involving cytosine methylation. However, few studies have focused on clarifying relationships between epigenetic influences of TEs on the adjacent DNA regions and time after insertion of TEs into the genome and/or their chromosomal location. Here we addressed these issues using soybean retrotransposon SORE-1. We analyzed SORE-1, inserted in exon 1 of the GmphyA2 gene, one of the newest insertions in this family so far identified. Cytosine methylation was detected in this element but was barely present in the adjacent regions. These results were correlated, respectively, with the presence and absence of the production of short interfering RNAs. Cytosine methylation profiles of 74 SORE-1 elements in the Williams 82 reference genome indicated that methylation frequency in the adjacent regions of SORE-1 was profoundly higher in pericentromeric regions than in euchromatic chromosome arms and was only weakly correlated with the length of time after insertion into the genome. Notably, the higher level of methylation in the 5' adjacent regions of SORE-1 coincided with the presence of repetitive elements in pericentromeric regions. Together, these results suggest that epigenetic influence of SORE-1 on the adjacent regions is influenced by its location on the chromosome.

Frequent generation of mutants with coincidental changes in multiple traits via ion-beam irradiation in soybean.

Ion beams are powerful mutagens that can induce novel mutants in plants. We previously established a system for producing a mutant population of soybean via ion-beam irradiation, isolated plants that had chlorophyll deficiency, and maintained their progeny via self-fertilization. Here we report the characterization of the progeny plants in terms of chlorophyll content, flowering time and isoflavone content in seeds. Chlorophyll deficiency in the leaf tissues was linked with reduced levels of isoflavones, the major flavonoid compounds accumulated in soybean seeds, which suggested the involvement of metabolic changes associated with the chlorophyll deficiency. Intriguingly, flowering time was frequently altered in plants that had a reduced level of chlorophyll in the leaf tissues. Plant lines that flowered either earlier or later than the wild-type plants were detected. The observed coincidental changes were presumed to be attributable to the following origins: structural changes of DNA segments leading to the loss of multiple gene functions, or indirect effects of mutations that affect one of these traits, which were manifested as phenotypic changes in the background of the duplicated composition of the soybean genome.

RNA silencing in the life cycle of soybean: multiple restriction systems and spatiotemporal variation associated with plant architecture.

The expression of transgenes introduced into a plant genome is sometimes suppressed by RNA silencing. Although local and systemic spread of RNA silencing have been studied, little is known about the mechanisms underlying spatial and temporal variation in transgene silencing between individual plants or between plants of different generations, which occurs seemingly stochastically. Here, we analyzed the occurrence, spread, and transmission of RNA silencing of the green fluorescent protein (GFP) gene over multiple generations of the progeny of a single soybean transformant. Observation of GFP fluorescence in entire plants of the T3-T5 generations indicated that the initiation and subsequent spread of GFP silencing varied between individuals, although this GFP silencing most frequently began in the primary leaves. In addition, GFP silencing could spread into the outer layer of seed coat tissues but was hardly detectable in the embryos. These results are consistent with the notion that transgene silencing involves its reset during reproductive phase, initiation after germination, and systemic spread in each generation. GFP silencing was absent in the pulvinus, suggesting that its cortical cells inhibit cell-to-cell spread or induction of RNA silencing. The extent of GFP silencing could differ between the stem and a petiole or between petiolules, which have limited vascular bundles connecting them and thus deter long-distant movement of silencing. Taken together, these observations indicate that the initiation and/or spread of RNA silencing depend on specific features of the architecture of the plant in addition to the mechanisms that can be conserved in higher plants.

Corrigendum: Enhancement of RNA-directed DNA methylation of a transgene by simultaneously downregulating a ROS1 ortholog using a virus vector in Nicotiana benthamiana.

[This corrects the article on p. 44 in vol. 4, PMID: 23565118.].

Corrigendum: Enhancement of RNA-directed DNA methylation of a transgene by simultaneously downregulating a ROS1 ortholog using a virus vector in Nicotiana benthamiana.

[This corrects the article on p. 44 in vol. 4, PMID: 23565118.].

The Soybean-Specific Maturity Gene E1 Family of Floral Repressors Controls Night-Break Responses through Down-Regulation of FLOWERING LOCUS T Orthologs.

Photoperiodism is a rhythmic change of sensitivity to light, which helps plants to adjust flowering time according to seasonal changes in daylength and to adapt to growing conditions at various latitudes. To reveal the molecular basis of photoperiodism in soybean (Glycine max), a facultative short-day plant, we analyzed the transcriptional profiles of the maturity gene E1 family and two FLOWERING LOCUS T (FT) orthologs (FT2a and FT5a). E1, a repressor for FT2a and FT5a, and its two homologs, E1-like-a (E1La) and E1Lb, exhibited two peaks of expression in long days. Using two different approaches (experiments with transition between light and dark phases and night-break experiments), we revealed that the E1 family genes were expressed only during light periods and that their induction after dawn in long days required a period of light before dusk the previous day. In the cultivar Toyomusume, which lacks the E1 gene, virus-induced silencing of E1La and E1Lb up-regulated the expression of FT2a and FT5a and led to early flowering. Therefore, E1, E1La, and E1Lb function similarly in flowering. Regulation of E1 and E1L expression by light was under the control of E3 and E4, which encode phytochrome A proteins. Our data suggest that phytochrome A-mediated transcriptional induction of E1 and its homologs by light plays a critical role in photoperiodic induction of flowering in soybean.

Erratum: In planta assays involving epigenetically silenced genes reveal inhibition of cytosine methylation by genistein.

[This corrects the article DOI: 10.1186/1746-4811-8-10.].

Induction of stable epigenetic gene silencing in plants using a virus vector.

Gene silencing through transcriptional repression can be induced by double-stranded RNA targeted to a gene promoter, a process known as RNA-mediated transcriptional gene silencing (TGS). This phenomenon is associated with epigenetic changes involving cytosine methylation of the promoter. Plant virus vectors have been used to induce RNA-mediated TGS. Here, we describe methods relevant to the induction of epigenetic changes and RNA-mediated TGS in plants using a virus vector, which include inoculation of recombinant virus, detection of short interfering RNAs, bisulfite sequencing analysis, and nuclear run-on transcription assay.

Enhancement of RNA-directed DNA methylation of a transgene by simultaneously downregulating a ROS1 ortholog using a virus vector in Nicotiana benthamiana.

Cytosine methylation can be induced by double-stranded RNAs through the RNA-directed DNA methylation (RdDM) pathway. A DNA glycosylase REPRESSOR OF SILENCING 1 (ROS1) participates in DNA demethylation in Arabidopsis and may possibly counteract RdDM. Here, we isolated an ortholog of ROS1 (NbROS1) from Nicotiana benthamiana and examined the antagonistic activity of NbROS1 against virus-induced RdDM by simultaneously inducing RdDM and NbROS1 knockdown using a vector based on Cucumber mosaic virus. Plants were inoculated with a virus that contained a portion of the Cauliflower mosaic virus 35S promoter, which induced RdDM of the promoter integrated in the plant genome and transcriptional silencing of the green fluorescent protein gene driven by the promoter. Plants were also inoculated with a virus that contained a portion of NbROS1, which induced downregulation of NbROS1. Simultaneous induction of RdDM and NbROS1 knockdown resulted in an increase in the level of cytosine methylation of the target promoter. These results provide evidence for the presence of antagonistic activity of NbROS1 against virus-induced RdDM and suggest that the simultaneous induction of promoter-targeting RdDM and NbROS1 knockdown by a virus vector is useful as a tool to enhance targeted DNA methylation.

Deep sequencing uncovers commonality in small RNA profiles between transgene-induced and naturally occurring RNA silencing of chalcone synthase-A gene in petunia.

BACKGROUND: Introduction of a transgene that transcribes RNA homologous to an endogenous gene in the plant genome can induce silencing of both genes, a phenomenon termed cosuppression. Cosuppression was first discovered in transgenic petunia plants transformed with the CHS-A gene encoding chalcone synthase, in which nonpigmented sectors in flowers or completely white flowers are produced. Some of the flower-color patterns observed in transgenic petunias having CHS-A cosuppression resemble those in existing nontransgenic varieties. Although the mechanism by which white sectors are generated in nontransgenic petunia is known to be due to RNA silencing of the CHS-A gene as in cosuppression, whether the same trigger(s) and/or pattern of RNA degradation are involved in these phenomena has not been known. Here, we addressed this question using deep-sequencing and bioinformatic analyses of small RNAs. RESULTS: We analyzed short interfering RNAs (siRNAs) produced in nonpigmented sectors of petal tissues in transgenic petunia plants that have CHS-A cosuppression and a nontransgenic petunia variety Red Star, that has naturally occurring CHS-A RNA silencing. In both silencing systems, 21-nt and 22-nt siRNAs were the most and the second-most abundant size classes, respectively. CHS-A siRNA production was confined to exon 2, indicating that RNA degradation through the RNA silencing pathway occurred in this exon. Common siRNAs were detected in cosuppression and naturally occurring RNA silencing, and their ranks based on the number of siRNAs in these plants were correlated with each other. Noticeably, highly abundant siRNAs were common in these systems. Phased siRNAs were detected in multiple phases at multiple sites, and some of the ends of the regions that produced phased siRNAs were conserved. CONCLUSIONS: The features of siRNA production found to be common to cosuppression and naturally occurring silencing of the CHS-A gene indicate mechanistic similarities between these silencing systems especially in the biosynthetic processes of siRNAs including cleavage of CHS-A transcripts and subsequent production of secondary siRNAs in exon 2. The data also suggest that these events occurred at multiple sites, which can be a feature of these silencing phenomena.

RNA silencing as a tool to uncover gene function and engineer novel traits in soybean.

RNA silencing refers collectively to diverse RNA-mediated pathways of nucleotide-sequence-specific inhibition of gene expression. It has been used to analyze gene function and engineer novel traits in various organisms. Here, we review the application of RNA silencing in soybean. To produce soybean lines, in which a particular gene is stably silenced, researchers have frequently used a transgene that transcribes inverted repeats of a target gene segment. Suppression of gene expression in developing soybean embryos has been one of the main focuses of metabolic engineering using transgene-induced silencing. Plants that have enhanced resistance against diseases caused by viruses or cyst nematode have also been produced. Meanwhile, Agrobacterium rhizogenes-mediated transformation has been used to induce RNA silencing in roots, which enabled analysis of the roles of gene products in nodulation or disease resistance. RNA silencing has also been induced using viral vectors, which is particularly useful for gene function analysis. So far, three viral vectors for virus-induced gene silencing have been developed for soybean. One of the features of the soybean genome is the presence of a large number of duplicated genes. Potential use of RNA silencing technology in combination with forward genetic approaches for analyzing duplicated genes is discussed.

In planta assays involving epigenetically silenced genes reveal inhibition of cytosine methylation by genistein.

BACKGROUND: Cytosine methylation is involved in epigenetic control of gene expression in a wide range of organisms. An increasing number of examples indicate that changing the frequency of cytosine methylation in the genome is a feasible tool to engineer novel traits in plants. Although demethylating effects of compounds have been analyzed in human cultured cells in terms of suppressing cancer, their effect in plant cells has not been analyzed extensively. Here, we developed in planta assay systems to detect inhibition of cytosine methylation using plants that contain a transgene transcriptionally silenced by an epigenetic mechanism. RESULTS: Seeds of two transgenic plants were used: a petunia line that has been identified as a revertant of the co-suppression of the chalcone synthase-A (CHS-A) gene and contains CHS-A transgenes whose transcription is repressed; Nicotiana benthamiana plants that contain the green fluorescent protein (GFP) reporter gene whose transcription is repressed through virus-induced transcriptional gene silencing. Seeds of these plants were sown on a medium that contained a demethylating agent, either 5-azacytidine or trichostatin A, and the restoration of the transcriptionally active state of the transgene was detected in seedlings. Using these systems, we found that genistein, a major isoflavonoid compound, inhibits cytosine methylation, thus restoring transgene transcription. Genistein also restored the transcription of an epigenetically silenced endogenous gene in Arabidopsis plants. CONCLUSIONS: Our assay systems allowed us to assess the inhibition of cytosine methylation, in particular of maintenance of methylation, by compounds in plant cells. These results suggest a novel role of flavonoids in plant cells and that genistein is useful for modifying the epigenetic state of plant genomes.

Coincident sequence-specific RNA degradation of linked transgenes in the plant genome.

The expression of transgenes in plant genomes can be inhibited by either transcriptional gene silencing or posttranscriptional gene silencing (PTGS). Overexpression of the chalcone synthase-A (CHS-A) transgene triggers PTGS of CHS-A and thus results in loss of flower pigmentation in petunia. We previously demonstrated that epigenetic inactivation of CHS-A transgene transcription leads to a reversion of the PTGS phenotype. Although neomycin phosphotransferase II (nptII), a marker gene co-introduced into the genome with the CHS-A transgene, is not normally silenced in petunia, even when CHS-A is silenced, here we found that nptII was silenced in a petunia line in which CHS-A PTGS was induced, but not in the revertant plants that had no PTGS of CHS-A. Transcriptional activity, accumulation of short interfering RNAs, and restoration of mRNA level after infection with viruses that had suppressor proteins of gene silencing indicated that the mechanism for nptII silencing was posttranscriptional. Read-through transcripts of the CHS-A gene toward the nptII gene were detected. Deep-sequencing analysis revealed a striking difference between the predominant size class of small RNAs produced from the read-through transcripts (22 nt) and that from the CHS-A RNAs (21 nt). These results implicate the involvement of read-through transcription and distinct phases of RNA degradation in the coincident PTGS of linked transgenes and provide new insights into the destabilization of transgene expression.

RNA-mediated epigenetic modifications of an endogenous gene targeted by a viral vector: a potent gene silencing system to produce a plant that does not carry a transgene but has altered traits.

Nucleotide-sequence-specific interactions mediated by double-stranded RNA (dsRNA) can induce gene silencing. Gene silencing through transcriptional repression can be induced by dsRNA targeted to a gene promoter. However, until recently, no plant has been produced that harbors an endogenous gene that remains silenced in the absence of promoter-targeting dsRNA. We have reported for the first time that transcriptional gene silencing can be induced by targeting dsRNA to the endogenous gene promoters in petunia and tomato plants, using a Cucumber mosaic virus (CMV)-based vector and that the induced gene silencing is heritable. Efficient silencing depended on the function of the 2b protein encoded in the vector, which facilitates epigenetic modifications through the transport of short interfering RNA (siRNA) to the nucleus. Here we show that gene silencing that is mediated by targeting dsRNA to a gene promoter via the CMV vector can be as strong as co-suppression in terms of both the extent of mRNA decrease and phenotypic changes. We also show that the expression of genes involved in RNA-directed DNA methylation and in demethylation are upregulated and downregulated, respectively, in Arabidopsis plants infected with CMV. Thus, along with the function of the 2b protein, that transports siRNA to the nucleus, the promoter-targeted silencing system using the CMV vector has some property that facilitates heritable epigenetic changes on endogenous genes, enabling the production of a novel class of modified plants that do not have a transgene.

Fertility restoration by Ifr1 in rice with BT-type cytoplasmic male sterility is associated with a reduced level, but not processing, of atp6-orf79 co-transcribed RNA.

BT-type cytoplasmic male sterility (CMS) in rice is associated with accumulation of unprocessed dicistronic RNA containing a duplicated atp6 (B-atp6) and an unusual open reading frame, orf79, encoding a cytotoxic peptide in mitochondria. The male-sterile state of BT-type CMS is stably maintained by backcrossing the plants with line Taichung 65 (T65) that has no restorer gene and is completely suppressed by the presence of the Rf1 gene through the processing of B-atp6-orf79 RNA. A variant of the T65 line, T65(T), has a weak restoration function conferred by the Ifr1 gene, which is genetically independent of the Rf1 gene. However, not much is known about the mechanism(s). In a study to examine whether the mechanism involved in fertility restoration by Ifr1 is analogous to restoration mediated by Rf1, the transcript profile of B-atp6-orf79 in male-sterile plants was compared with that in fertility restored plants obtained by crossing male-sterile plants with T65(T). The cellular level of unprocessed B-atp6-orf79 RNA was reduced in the restored plants, but no change in processing efficiency or the quantity of B-atp6-orf79 DNA was detected. These results suggest that Ifr1 restores fertility through reducing either the transcription rate of B-atp6-orf79 or the stability of its primary transcripts, a mechanism distinct from that involved in fertility restoration of BT-type CMS by Rf1.