Hidetaka Ito

The genome sequence and structure of rice chromosome 1

The rice species Oryza sativa is considered to be a model plant because of its small genome size, extensive genetic map, relative ease of transformation and synteny with other cereal crops. Here we report the essentially complete sequence of chromosome 1, the longest chromosome in the rice genome. We summarize characteristics of the chromosome structure and the biological insight gained from the sequence. The analysis of 43.3 megabases (Mb) of non-overlapping sequence reveals 6,756 protein coding genes, of which 3,161 show homology to proteins of Arabidopsis thaliana, another model plant. About 30% (2,073) of the genes have been functionally categorized. Rice chromosome 1 is (G + C)-rich, especially in its coding regions, and is characterized by several gene families that are dispersed or arranged in tandem repeats. Comparison with a draft sequence indicates the importance of a high-quality finished sequence.

An siRNA pathway prevents transgenerational retrotransposition in plants subjected to stress

Eukaryotic genomes consist to a significant extent of retrotransposons that are suppressed by host epigenetic mechanisms, preventing their uncontrolled propagation. However, it is not clear how this is achieved. Here we show that in Arabidopsis seedlings subjected to heat stress, a copia-type retrotransposon named ONSEN (Japanese ‘hot spring’) not only became transcriptionally active but also synthesized extrachromosomal DNA copies. Heat-induced ONSEN accumulation was stimulated in mutants impaired in the biogenesis of small interfering RNAs (siRNAs); however, there was no evidence of transposition occurring in vegetative tissues. After stress, both ONSEN transcripts and extrachromosomal DNA gradually decayed and were no longer detected after 20–30 days. Surprisingly, a high frequency of new ONSEN insertions was observed in the progeny of stressed plants deficient in siRNAs. Insertion patterns revealed that this transgenerational retrotransposition occurred during flower development and before gametogenesis. Therefore in plants with compromised siRNA biogenesis, memory of stress was maintained throughout development, priming ONSEN to transpose during differentiation of generative organs. Retrotransposition was not observed in the progeny of wild-type plants subjected to stress or in non-stressed mutant controls, pointing to a crucial role of the siRNA pathway in restricting retrotransposition triggered by environmental stress. Finally, we found that natural and experimentally induced variants in ONSEN insertions confer heat responsiveness to nearby genes, and therefore mobility bursts may generate novel, stress-responsive regulatory gene networks.

Genomic and Genetic Characterization of Rice Cen3 Reveals Extensive Transcription and Evolutionary Implications of a Complex Centromere

The centromere is the chromosomal site for assembly of the kinetochore where spindle fibers attach during cell division. In most multicellular eukaryotes, centromeres are composed of long tracts of satellite repeats that are recalcitrant to sequencing and fine-scale genetic mapping. Here, we report the genomic and genetic characterization of the complete centromere of rice (Oryza sativa) chromosome 3. Using a DNA fiber-fluorescence in situ hybridization approach, we demonstrated that the centromere of chromosome 3 (Cen3) contains ∼441 kb of the centromeric satellite repeat CentO. Cen3 includes an ∼1,881-kb domain associated with the centromeric histone CENH3. This CENH3-associated chromatin domain is embedded within a 3113-kb region that lacks genetic recombination. Extensive transcription was detected within the CENH3 binding domain based on comprehensive annotation of protein-coding genes coupled with empirical measurements of mRNA levels using RT-PCR and massively parallel signature sequencing. Genes <10 kb from the CentO satellite array were expressed in several rice tissues and displayed histone modification patterns consistent with euchromatin, suggesting that rice centromeric chromatin accommodates normal gene expression. These results support the hypothesis that centromeres can evolve from gene-containing genomic regions.

The effects of heat induction and the siRNA biogenesis pathway on the transgenerational transposition of ONSEN, a copia-like retrotransposon in Arabidopsis thaliana

Environmental stress influences genetic and epigenetic regulation in plant genomes. We previously reported that heat stress activated a copia-like retrotransposon named ONSEN. To investigate the heat sensitivity and transgenerational activation of ONSEN, we analyzed the stress response by temperature shift and multiple heat stress treatments. ONSEN was activated at 37°C, and the newly inserted ONSEN was transcriptionally active and mobile to the next generation subjected to heat stress, indicating that the regulation of ONSEN is independent of positional effects on the chromosome. Reciprocal crosses with activated ONSEN revealed that the transgenerational transposition was inherited from both sexes, indicating that the transposition is suppressed independently of gametophytic regulation. We showed previously that ONSEN was transposed in mutants deficient in small interfering RNA (siRNA) biogenesis, including nrpd2 and rdr2, but not dcl3. To define the functional redundancy of Dicer-like (DCL) proteins in Arabidopsis, we analyzed ONSEN activation in mutants deficient in DCL proteins, including dcl2, dcl3 and dcl4. ONSEN was nearly immobile in a single Dicer mutant; however, some transgenerational transpositions were observed in dcl2/dcl3/dcl4 triple mutants subjected to heat stress. This indicated that the Dicer family is redundant for ONSEN transposition. To examine the activation of ONSEN in undifferentiated cells, ONSEN transcripts and synthesized DNA were analyzed in heat-stressed callus tissue. In contrast to vegetative tissue, high accumulation of the transcripts and amplified DNA copies of ONSEN were detected in callus. This result indicated that ONSEN activation is controlled by cell-specific regulatory mechanisms.

A Stress-Activated Transposon in Arabidopsis Induces Transgenerational Abscisic Acid Insensitivity.

Transposable elements (TEs), or transposons, play an important role in adaptation. TE insertion can affect host gene function and provides a mechanism for rapid increases in genetic diversity, particularly because many TEs respond to environmental stress. In the current study, we show that the transposition of a heat-activated retrotransposon, ONSEN, generated a mutation in an abscisic acid (ABA) responsive gene, resulting in an ABA-insensitive phenotype in Arabidopsis, suggesting stress tolerance. Our results provide direct evidence that a transposon activated by environmental stress could alter the genome in a potentially positive manner. Furthermore, the ABA-insensitive phenotype was inherited when the transcription was disrupted by an ONSEN insertion, whereas ABA sensitivity was recovered when the effects of ONSEN were masked by IBM2. These results suggest that epigenetic mechanisms in host plants typically buffered the effect of a new insertion, but could selectively “turn on” TEs when stressed.

Evolution of the ONSEN retrotransposon family activated upon heat stress in Brassicaceae

A Ty1/Copia-like retrotransposon, ONSEN, is activated by heat stress in Arabidopsis thaliana, and its de novo integrations that were observed preferentially within genes implies its regulation of neighboring genes. Here we show that ONSEN related copies were found in most species of Brassicaceae, forming a cluster with each species in phylogenetic tree. Most copies were localized close to genes in Arabidopsis lyrata and Brassica rapa, suggesting conserved integration specificity of ONSEN family into genic or open chromatin. In addition, we found heat-induced transcriptional activation of ONSEN family in several species of Brassicaceae. These results suggest that ONSEN has conserved transcriptional activation promoted by environmental heat stress in some Brassicaceae species.

Control of transposable elements in Arabidopsis thaliana

Arabidopsis thaliana serves as a very good model organism to investigate the control of transposable elements (TEs) by genetic and genomic approaches. As TE movements are potentially deleterious to the hosts, hosts silence TEs by epigenetic mechanisms, such as DNA methylation. DNA methylation is controlled by DNA methyltransferases and other regulators, including histone modifiers and chromatin remodelers. RNAi machinery directs DNA methylation to euchromatic TEs, which is under developmental control. In addition to the epigenetic controls, some TEs are controlled by environmental factors. TEs often affect expression of nearby genes, providing evolutionary sources for epigenetic, developmental, and environmental gene controls, which could even be beneficial for the host.

Small RNAs and transposon silencing in plants

Transposons are highly conserved in plants and have created a symbiotic relationship with the host genome. An important factor of the successful communication between transposons and host plants is epigenetic modifications including DNA methylation and the modifications of the histone tail. In plants, small interfering RNAs (siRNAs) are responsible for RNA‐directed DNA methylation (RdDM) that suppresses transposon activities. Although most transposons are silent in their host plants, certain genomic shocks, such as an environmental stress or a hybridization event, might trigger transposon activation. Further, since transposons can affect the regulation mechanisms of host genes, it is possible that transposons have co‐evolved as an important mechanism for plant development and adaptation. Recent new findings reveal that siRNAs control not only transcriptional activation, but also suppress transgenerational transposition of mobile elements making siRNAs critically important towards maintaining genome stability. Together these data suggest host‐mediated siRNA regulation of transposons appears to have been adapted for controlling essential systems of plant development, morphogenesis, and reproduction.

A small RNA mediated regulation of a stress-activated retrotransposon and the tissue specific transposition during the reproductive period in Arabidopsis

Transposable elements (TEs) are key elements that facilitate genome evolution of the host organism. A number of studies have assessed the functions of TEs, which change gene expression in the host genome. Activation of TEs is controlled by epigenetic modifications such as DNA methylation and histone modifications. Several recent studies have reported that TEs can also be activated by biotic or abiotic stress in some plants. We focused on a Ty1/copia retrotransposon, ONSEN, that is activated by heat stress (HS) in Arabidopsis. We found that transcriptional activation of ONSEN was regulated by a small interfering RNA (siRNA)-related pathway, and the activation could also be induced by oxidative stress. Mutants deficient in siRNA biogenesis that were exposed to HS at the initial stages of vegetative growth showed transgenerational transposition. The transposition was also detected in the progeny, which originated from tissue that had differentiated after exposure to the HS. The results indicated that in some undifferentiated cells, transpositional activity could be maintained quite long after exposure to the HS.

Overexpression of the TIR-X gene results in a dwarf phenotype and activation of defense-related gene expression in Arabidopsis thaliana.

The Arabidopsis genome encodes various proteins with a Toll/interleukin-1 receptor (TIR) domain. Many of these proteins also contain nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domains and function as resistance (R) proteins. However, the protein encoded by At2g32140 (a TIR-X gene) contains a TIR domain but lacks NBS and LRR domains. We found that transgenic plants overexpressing At2g32140 displayed a dwarf phenotype and showed increased expression of defense-related genes. In general, the growth defect caused by activation of defense responses is suppressed under high-temperature conditions. However, transgenic plants overexpressing At2g32140 displayed a much stronger dwarf phenotype at 28°C than at 22°C. This dwarf phenotype was suppressed under the combination with known salicylic-acid pathway mutants. These findings suggest that At2g32140 encodes a protein involved in the plant defense response.