Akie Kobayashi

Bursts of retrotransposition reproduced in Arabidopsis

Retrotransposons, which proliferate by reverse transcription of RNA intermediates, comprise a major portion of plant genomes. Plants often change the genome size and organization during evolution by rapid proliferation and deletion of long terminal repeat (LTR) retrotransposons. Precise transposon sequences throughout the Arabidopsis thaliana genome and the trans-acting mutations affecting epigenetic states make it an ideal model organism with which to study transposon dynamics. Here we report the mobilization of various families of endogenous A. thaliana LTR retrotransposons identified through genetic and genomic approaches with high-resolution genomic tiling arrays and mutants in the chromatin-remodelling gene DDM1 (DECREASE IN DNA METHYLATION 1). Using multiple lines of self-pollinated ddm1 mutant, we detected an increase in copy number, and verified this for various retrotransposons in a gypsy family (ATGP3) and copia families (ATCOPIA13, ATCOPIA21, ATCOPIA93), and also for a DNA transposon of a Mutator family, VANDAL21. A burst of retrotransposition occurred stochastically and independently for each element, suggesting an additional autocatalytic process. Furthermore, comparison of the identified LTR retrotransposons in related Arabidopsis species revealed that a lineage-specific burst of retrotransposition of these elements did indeed occur in natural Arabidopsis populations. The recent burst of retrotransposition in natural population is targeted to centromeric repeats, which is presumably less harmful than insertion into genes. The ddm1-induced retrotransposon proliferations and genome rearrangements mimic the transposon-mediated genome dynamics during evolution and provide experimental systems with which to investigate the controlling molecular factors directly.

An Arabidopsis jmjC domain protein protects transcribed genes from DNA methylation at CHG sites

Differential cytosine methylation of genes and transposons is important for maintaining integrity of plant genomes. In Arabidopsis, transposons are heavily methylated at both CG and non‐CG sites, whereas the non‐CG methylation is rarely found in active genes. Our previous genetic analysis suggested that a jmjC domain‐containing protein IBM1 (increase in BONSAI methylation 1) prevents ectopic deposition of non‐CG methylation, and this process is necessary for normal Arabidopsis development. Here, we directly determined the genomic targets of IBM1 through high‐resolution genome‐wide analysis of DNA methylation. The ibm1 mutation induced extensive hyper‐methylation in thousands of genes. Transposons were unaffected. Notably, long transcribed genes were most severely affected. Methylation of genes is limited to CG sites in wild type, but CHG sites were also methylated in the ibm1 mutant. The ibm1‐induced hyper‐methylation did not depend on previously characterized components of the RNAi‐based DNA methylation machinery. Our results suggest novel transcription‐coupled mechanisms to direct genic methylation not only at CG but also at CHG sites. IBM1 prevents the CHG methylation in genes, but not in transposons.

A gene essential for hydrotropism in roots

Roots display hydrotropism in response to moisture gradients, which is thought to be important for controlling their growth orientation, obtaining water, and establishing their stand in the terrestrial environment. However, the molecular mechanism underlying hydrotropism remains unknown. Here, we report that roots of the Arabidopsis mutant mizu-kussei1 (miz1), which are impaired in hydrotropism, show normal gravitropism and elongation growth. The roots of miz1 plants showed reduced phototropism and a modified wavy growth response. There were no distinct differences in morphological features and root structure between miz1 and wild-type plants. These results suggest that the pathway inducing hydrotropism is independent of the pathways used in other tropic responses. The phenotype results from a single recessive mutation in MIZ1, which encodes a protein containing a domain (the MIZ domain) that is highly conserved among terrestrial plants such as rice and moss. The MIZ domain was not found in known genomes of organisms such as green algae, red algae, cyanobacteria, or animals. We hypothesize that MIZ1 has evolved to play an important role in adaptation to terrestrial life because hydrotropism could contribute to drought avoidance in higher plants. In addition, a pMIZ1::GUS fusion gene was expressed strongly in columella cells of the root cap but not in the elongation zone, suggesting that MIZ1 functions in the early phase of the hydrotropic response.

Hydrotropism Interacts with Gravitropism by Degrading Amyloplasts in Seedling Roots of Arabidopsis and Radish

In response to a moisture gradient, roots exhibit hydrotropism to control the orientation of their growth. To exhibit hydrotropism, however, they must overcome the gravitropism that is dominant on Earth. We found that moisture gradient or water stress caused immediate degradation of the starch anchors, amyloplasts, in root columella cells of Arabidopsis and radish (Raphanus sativus). Namely, development of hydrotropic response was accompanied by a simultaneous reduction in starch content in columella cells. Rapid degradation of amyloplasts in columella cells also occurred in the water-stressed roots with sorbitol or mannitol. Both hydrotropically stimulated and water-stressed roots showed a reduced responsiveness to gravity. Roots of a starchless mutant, pgm1-1, showed an enhanced hydrotropism compared with that of the wild type. These results suggest that the reduced responsiveness to gravity is, at least in part, attributable to the degradation of amyloplasts in columella cells. Thus, the reduction in gravitropism allows the roots to exhibit hydrotropism.

GNOM-Mediated Vesicular Trafficking Plays an Essential Role in Hydrotropism of Arabidopsis Roots

Roots respond not only to gravity but also to moisture gradient by displaying gravitropism and hydrotropism, respectively, to control their growth orientation, which helps plants obtain water and become established in the terrestrial environment. As gravitropism often interferes with hydrotropism, however, the mechanisms of how roots display hydrotropism and differentiate it from gravitropism are not understood. We previously reported MIZU-KUSSEI1 (MIZ1) as a gene required for hydrotropism but not for gravitropism, although the function of its protein was not known. Here, we found that a mutation of GNOM encoding guanine-nucleotide exchange factor for ADP-ribosylation factor-type G proteins was responsible for the ahydrotropism of Arabidopsis (Arabidopsis thaliana), miz2. Unlike other gnom alleles, miz2 showed no apparent morphological defects or reduced gravitropism. Instead, brefeldin A (BFA) treatment inhibited both hydrotropism and gravitropism in Arabidopsis roots. In addition, a BFA-resistant GNOM variant, GNM696L, showed normal hydrotropic response in the presence of BFA. Furthermore, a weak gnom allele, gnomB/E, showed defect in hydrotropic response. These results indicate that GNOM-mediated vesicular trafficking plays an essential role in hydrotropism of seedling roots.

Centromere-targeted de novo integrations of an LTR retrotransposon of Arabidopsis lyrata

The plant genome evolves with rapid proliferation of LTR-type retrotransposons, which is associated with their clustered accumulation in gene-poor regions, such as centromeres. Despite their major role for plant genome evolution, no mobile LTR element with targeted integration into gene-poor regions has been identified in plants. Here, we report such targeted integrations de novo. We and others have previously shown that an ATCOPIA93 family retrotransposon in Arabidopsis thaliana is mobilized when the DNA methylation machinery is compromised. Although ATCOPIA93 family elements are low copy number in the wild-type A. thaliana genome, high-copy-number related elements are found in the wild-type Arabidopsis lyrata genome, and they show centromere-specific localization. To understand the mechanisms for the clustered accumulation of the A. lyrata elements directly, we introduced one of them, named Tal1 (Transposon of Arabidopsis lyrata 1), into A. thaliana by transformation. The introduced Tal1 was retrotransposed in A. thaliana, and most of the retrotransposed copies were found in centromeric repeats of A. thaliana, suggesting targeted integration. The targeted integration is especially surprising because the centromeric repeat sequences differ considerably between A. lyrata and A. thaliana. Our results revealed unexpectedly dynamic controls for evolution of the transposon-rich heterochromatic 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.

Molecular mechanisms of hydrotropism in seedling roots of Arabidopsis thaliana (Brassicaceae)

Roots show positive hydrotropism in response to moisture gradients, which is believed to contribute to plant water acquisition. This article reviews the recent advances of the physiological and molecular genetic studies on hydrotropism in seedling roots of Arabidopsis thaliana. We identified MIZU-KUSSEI1 (MIZ1) and MIZ2, essential genes for hydrotropism in roots; the former encodes a protein of unknown function, and the latter encodes an ARF-GEF (GNOM) protein involved in vesicle trafficking. Because both mutants are defective in hydrotropism but not in gravitropism, these mutations might affect a molecular mechanism unique to hydrotropism. MIZ1 is expressed in the lateral root cap and cortex of the root proper. It is localized as a soluble protein in the cytoplasm and in association with the cytoplasmic face of endoplasmic reticulum (ER) membranes in root cells. Light and ABA independently regulate MIZ1 expression, which influences the ultimate hydrotropic response. In addition, MIZ1 overexpression results in an enhancement of hydrotropism and an inhibition of lateral root formation. This phenotype is likely related to the alteration of auxin content in roots. Specifically, the auxin level in the roots decreases in the MIZ1 overexpressor and increases in the miz1 mutant. Unlike most gnom mutants, miz2 displays normal morphology, growth, and gravitropism, with normal localization of PIN proteins. It is probable that MIZ1 plays a crucial role in hydrotropic response by regulating the endogenous level of auxin in Arabidopsis roots. Furthermore, the role of GNOM/MIZ2 in hydrotropism is distinct from that of gravitropism.

Hormonal Regulation of Lateral Root Development in Arabidopsis Modulated by MIZ1 and Requirement of GNOM Activity for MIZ1 Function

Plant organ development is important for adaptation to a changing environment. Genetic and physiological studies have revealed that plant hormones play key roles in lateral root formation. In this study, we show that MIZU-KUSSEI1 (MIZ1), which was identified originally as a regulator of hydrotropism, functions as a novel regulator of hormonally mediated lateral root development. Overexpression of MIZ1 (MIZ1OE) in roots resulted in a reduced number of lateral roots being formed; however, this defect could be recovered with the application of auxin. Indole-3-acetic acid quantification analyses showed that free indole-3-acetic acid levels decreased in MIZ1OE roots, which indicates that alteration of auxin level is critical for the inhibition of lateral root formation in MIZ1OE plants. In addition, MIZ1 negatively regulates cytokinin sensitivity on root development. Application of cytokinin strongly induced the localization of MIZ1-green fluorescent protein to lateral root primordia, which suggests that the inhibition of lateral root development by MIZ1 occurs downstream of cytokinin signaling. Surprisingly, miz2, a weak allele of gnom, suppressed developmental defects in MIZ1OE plants. Taken together, these results suggest that MIZ1 plays a role in lateral root development by maintaining auxin levels and that its function requires GNOM activity. These data provide a molecular framework for auxin-dependent organ development in Arabidopsis (Arabidopsis thaliana).

RNAi‐independent de novo DNA methylation revealed in Arabidopsis mutants of chromatin remodeling gene DDM1

Methylation of histone H3 lysine 9 (H3K9me) and small RNAs are associated with constitutively silent chromatin in diverse eukaryotes including plants. In plants, silent transposons are also marked by cytosine methylation, especially at non-CpG sites. Transposon-specific non-CpG methylation in plants is controlled by small RNAs and H3K9me. Although it is often assumed that small RNA directs H3K9me, interaction between small RNA and H3K9me has not been directly demonstrated in plants. We have previously shown that a mutation in the chromatin remodeling gene DDM1 (DECREASE IN DNA METHYLATION 1) induces a global decrease but a local increase of cytosine methylation and accumulation of small RNA at a locus called BONSAI. Here we show that de novo BONSAI methylation does not depend on RNAi but does depend on H3K9me. In mutants of H3K9 methyltransferase gene KRYPTONITE or the H3K9me-dependent DNA methyltransferase gene CHROMOMETHYALSE3, the ddm1-induced de novo cytosine methylation was abolished for all three contexts (CpG, CpHpG and CpHpH). Furthermore, RNAi mutants showed strong developmental defects when combined with the ddm1 mutation. Our results revealed unexpected interactions of epigenetic modifications that may be conserved among diverse eukaryotes.