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In eukaryotes, homologous recombination plays a pivotal role in both genome maintenance and generation of genetic diversity. Eukaryotic RecA homologues, RAD51 and DMC1, are key proteins in homologous recombination that promote pairing between homologous DNA sequences. Arabidopsis thaliana is a prominent model plant for studying eukaryotic homologous recombination. However, A. thaliana RAD51 and DMC1 have not been biochemically characterized. In the present study, we purified A. thaliana RAD51 (AtRAD51) and DMC1 (AtDMC1). Biochemical analyses revealed that both AtRAD51 and AtDMC1 possess ATP hydrolyzing activity, filament formation activity and homologous pairing activity in vitro. We then compared the homologous pairing activities of AtRAD51 and AtDMC1 with those of the Oryza sativa and Homo sapiens RAD51 and DMC1 proteins.

Sulfoglycolipid, SQAP, is a radiosensitizing agent that makes tumor cells more sensitive to radiation therapy. A previous study revealed that SQAP induced the degradation of hypoxia-inducible factor-1α (HIF-1α) and inhibited angiogenesis in a hepatoma model mouse. Herein, we examined the biological activities of SQAP against hepatocarcinoma cells under low oxygen conditions. Cell growth inhibition of SQAP under hypoxic conditions was significantly higher than that under normoxic conditions. In addition, SQAP was found to impair the expression of histone deacetylase (HDAC) under low oxygen conditions. Our present data suggested that SQAP induced the degradation of HIF-1α and then decreased the expression of HDAC1. Unlike known HDAC inhibitors, SQAP increased the acetylation level of histone in cells without inhibition of enzymatic activity of HDACs. Our data demonstrated hypoxia-specific unique properties of SQAP.

Thiazoline-related innate fear-eliciting compounds (tFOs) orchestrate hypothermia, hypometabolism, and anti-hypoxia, which enable survival in lethal hypoxic conditions. Here, we show that most of these effects are severely attenuated in transient receptor potential ankyrin 1 (Trpa1) knockout mice. TFO-induced hypothermia involves the Trpa1-mediated trigeminal/vagal pathways and non-Trpa1 olfactory pathway. TFOs activate Trpa1-positive sensory pathways projecting from trigeminal and vagal ganglia to the spinal trigeminal nucleus (Sp5) and nucleus of the solitary tract (NTS), and their artificial activation induces hypothermia. TFO presentation activates the NTS-Parabrachial nucleus pathway to induce hypothermia and hypometabolism; this activation was suppressed in Trpa1 knockout mice. TRPA1 activation is insufficient to trigger tFO-mediated anti-hypoxic effects; Sp5/NTS activation is also necessary. Accordingly, we find a novel molecule that enables mice to survive in a lethal hypoxic condition ten times longer than known tFOs. Combinations of appropriate tFOs and TRPA1 command intrinsic physiological responses relevant to survival fate., Matsuo et al. report that thiazoline-related innate fear-eliciting compounds activate the spinal trigeminal nucleus (Sp5) and the nucleus of the solitary tract (NTS) via vagal/trigeminal TRPA1 to induce robust physiological alterations, enabling long time survival in a lethal hypoxic environment.

Visualization of intracellular structures and their spatial organization inside cells without any modification is essential to understand the mechanisms underlying the biological functions of cells. Here, we investigated the intracellular structure of cyanobacteria Prochlorococcus in the interphase by X-ray diffraction imaging using X-ray free-electron laser. A number of diffraction patterns from single cells smaller than 1 µm in size were collected with high signal-to-noise ratio with a resolution of up to 30 nm. From diffraction patterns, a set of electron density maps projected along the direction of the incident X-ray were retrieved with high reliability. The most characteristic structure found to be common among the cells was a C-shaped arrangement of 100-nm sized high-density spots, which surrounded a low-density area of 100 nm. Furthermore, a three-dimensional map reconstructed from the projection maps of individual cells was non-uniform, indicating the presence of common structures among cyanobacteria cells in the interphase. By referring to the fluorescent images for distributions of thylakoid membranes, nucleoids, and carboxysomes, we inferred and represented their spatial arrangements in the three-dimensional map. The arrangement allowed us to discuss the relevance of the intracellular organization to the biological functions of cyanobacteria.

Tissue clearing methods are increasingly essential for the microscopic observation of internal tissues of thick biological organs. We previously developed TOMEI, a clearing method for plant tissues; however, it could not entirely remove chlorophylls nor reduce the fluorescent signal of fluorescent proteins. Here, we developed an improved TOMEI method (iTOMEI) to overcome these limitations. First, a caprylyl sulfobetaine was determined to efficiently remove chlorophylls from Arabidopsis thaliana seedlings without GFP quenching. Next, a weak alkaline solution restored GFP fluorescence, which was mainly lost during fixation, and an iohexol solution with a high refractive index increased sample transparency. These procedures were integrated to form iTOMEI. iTOMEI enables the detection of much brighter fluorescence than previous methods in tissues of A. thaliana, Oryza sativa, and Marchantia polymorpha. Moreover, a mouse brain was also efficiently cleared by the iTOMEI-Brain method within 48 h, and strong fluorescent signals were detected in the cleared brain., Sakamoto et al. demonstrate an improved optical clearing method, iTOMEI, for plant imaging. The new method can achieve fast clearing and effective removal of autofluorescence signals, and at the same time preserve signals from desired fluorescence proteins.

The mitochondrion is an organelle that was derived from an endosymbiosis. Although regulation of mitochondrial growth by the host cell is necessary for the maintenance of mitochondria, it is unclear how this regulatory mechanism was acquired. To address this, we studied the primitive unicellular red alga Cyanidioschyzon merolae, which has the simplest eukaryotic genome and a single mitochondrion. Here we show that the C. merolae Aurora kinase ortholog CmAUR regulates mitochondrial division through phosphorylation of mitochondrial division ring components. One of the components, the Drp1 ortholog CmDnm1, has at least four sites phosphorylated by CmAUR. Depletion of the phosphorylation site conserved among eukaryotes induced defects such as mitochondrial distribution on one side of the cell. Taken together with the observation that human Aurora kinase phosphorylates Drp1 in vitro, we suggest that the phosphoregulation is conserved from the simplest eukaryotes to mammals, and was acquired at the primitive stage of endosymbiosis., Kato et al. show that red alga Cyanidioschyzon merolae aurora kinase CmAUR regulates mitochondrial division by phosphorylating mitochondrial division ring components. Depletion of the evolutionarily conserved phosphorylation site on CmAUR targets causes defective mitochondrial divisions, suggesting an ancient origin of this regulation during endosymbiosis.

Ion beams have been used as an effective tool in mutation breeding for the creation of crops with novel characteristics. Recent analyses have revealed that ion beams induce large chromosomal alterations, in addition to small mutations comprising base changes or frameshifts. In an effort to understand the potential capability of ion beams, we analyzed an Arabidopsis mutant possessing an abnormal genetic trait. The Arabidopsis mutant uvh3-2 is hypersensitive to UVB radiation when photoreactivation is unavailable. uvh3-2 plants grow normally and produce seeds by self-pollination. SSLP and CAPS analyses of F2 plants showed abnormal recombination frequency on chromosomes 2 and 3. PCR-based analysis and sequencing revealed that one-third of chromosome 3 was translocated to chromosome 2 in uvh3-2. FISH analysis using a 180 bp centromeric repeat and 45S ribosomal DNA (rDNA) as probes showed that the 45S rDNA signal was positioned away from that of the 180 bp centromeric repeat in uvh3-2, suggesting the insertion of a large chromosome fragment into the chromosome with 45S rDNA clusters. F1 plants derived from a cross between uvh3-2 and wild-type showed reduced fertility. PCR-based analysis of F2 plants suggested that reproductive cells carrying normal chromosome 2 and uvh3-2–derived chromosome 3 are unable to survive and therefore produce zygote. These results showed that ion beams could induce marked genomic alterations, and could possibly lead to the generation of novel plant species and crop strains.

Leaf color is an important indicator when evaluating plant growth and responses to biotic/abiotic stress. Acquisition of images by digital cameras allows analysis and long-term storage of the acquired images. However, under field conditions, where light intensity can fluctuate and other factors (shade, reflection, and background, etc.) vary, stable and reproducible measurement and quantification of leaf color are hard to achieve. Digital scanners provide fixed conditions for obtaining image data, allowing stable and reliable comparison among samples, but require detached plant materials to capture images, and the destructive processes involved often induce deformation of plant materials (curled leaves and faded colors, etc.). In this study, by using a lightweight digital scanner connected to a mobile computer, we obtained digital image data from intact plant leaves grown in natural-light greenhouses without detaching the targets. We took images of soybean leaves infected by Xanthomonas campestris pv. glycines, and distinctively quantified two disease symptoms (brown lesions and yellow halos) using freely available image processing software. The image data were amenable to quantitative and statistical analyses, allowing precise and objective evaluation of disease resistance.

International audience; Homologous recombination is a key process for maintaining genome integrity and diversity. In eukaryotes, the nucleosome structure of chromatin inhibits the progression of homologous recombination. The DNA repair and recombination protein RAD54 alters the chromatin structure via nucleosome sliding to enable homology searches. For homologous recombination to progress, appropriate recruitment and dissociation of RAD54 is required at the site of homologous recombination; however, little is known about the mechanism regulating RAD54 dynamics in chromatin. Here, we reveal that the histone demethylase LYSINE-SPECIFIC DEMETHYLASE1-LIKE 1 (LDL1) regulates the dissociation of RAD54 at damaged sites during homologous recombination repair in the somatic cells of Arabidopsis (Arabidopsis thaliana). Depletion of LDL1 leads to an overaccumulation of RAD54 at damaged sites with DNA double-strand breaks. Moreover, RAD54 accumulates at damaged sites by recognizing histone H3 Lys 4 di-methylation (H3K4me2); the frequency of the interaction between RAD54 and H3K4me2 increased in the ldl1 mutant with DNA double-strand breaks. We propose that LDL1 removes RAD54 at damaged sites by demethylating H3K4me2 during homologous recombination repair and thereby maintains genome stability in Arabidopsis.

As a sessile organism, plants are constantly challenged by diverse environmental stresses that threaten genome integrity by way of induction of DNA damage. In plants, each tissue is composed of differentiated cell types, and the response to DNA damage differs among each cell type. However, limited information is available on the subnuclear dynamics of different cell types in response to DNA damage in plants. A chromatin remodeling factor RAD54, which plays an important role in the exchange reaction and alteration of chromatin structure during homologous recombination, specifically accumulates at damaged sites, forming DNA repair foci (termed RAD54 foci) in nuclei after γ-irradiation. In this study, we performed a time-course analysis of the appearance of RAD54 foci in root cells of Arabidopsis after γ-irradiation to characterize the subnuclear dynamics in each cell type. A short time after γ-irradiation, no significant difference in detection frequency of RAD54 foci was observed among epidermal, cortical, and endodermal cells in the meristematic zone of roots. Interestingly, cells showing RAD54 foci persisted in roots at long time after γ-irradiation, and RAD54 foci in these cells localized to nuclear periphery with high frequency. These observations suggest that the nuclear envelope plays a role in the maintenance of genome stability in response to DNA damage in Arabidopsis roots.

Leaf development in plants, including dorsoventral (adaxial–abaxial) patterning, is tightly regulated. The involvement of several subunits of the 26S proteasome in adaxial–abaxial polarity establishment has been reported. In the present study, we revealed that in Arabidopsis thaliana, a mutation in RPT5A, a subunit of 26S proteasome, causes abnormally narrow true leaves under zinc deficiency. mRNA accumulations of DNA damage marker genes in leaves were elevated by zinc deficiency. PARP2, a single-strand break (SSB) inducible gene, was more strongly induced by zinc deficiency in rpt5a mutants compared with the wild type. A comet assay indicated that SSB is enhanced in mutants grown under the zinc deficiency condition. These results suggest that SSB accumulation is accompanied by abnormal leaf development. To test if DNA damage is a sole cause of abnormal leaf development, we treated the wild type grown under normal zinc conditions with zeocin, a DNA damage-inducing reagent, and found that narrow leaves developed, suggesting that DNA damage is sufficient to induce the development of abnormally narrow leaves. Taken together with the observation of the abnormal leaf morphology of our mutant plant under zinc deficiency, we demonstrated that the alleviation of DNA damage is important for normal leaf development.

Plants possess the structural maintenance of chromosome (SMC) protein complexes cohesin, condensin, and SMC5/6, which function in fundamental biological processes such as sister chromatid cohesion, chromosome condensation and segregation, and damaged DNA repair. Recently, increasing evidence in several organisms has suggested that condensin is involved in chromatin organizations during interphase. In Arabidopsis thaliana, condensin II is localized in the nucleus throughout interphase and is suggested to be required for keeping centromeres apart and the assembly of euchromatic chromosome arms. However, it remains unclear how condensin II organizes chromatin associations. Here, we first showed the high possibility that the function of condensin II as a complex is required for the disassociation of centromeres. Analysis of the rDNA array distribution revealed that condensin II is also indispensable for the association of centromeres with rDNA arrays. Reduced axial compaction of chromosomes and impaired genome integrity in condensin II mutants are not related to the disruption of chromatin organization. In contrast, the axial compaction of chromosomes by condensin II produces the force leading to the disassociation of heterologous centromeres in Drosophila melanogaster. Taken together, our data imply that the condensin II function in chromatin organization differs among eukaryotes.

The maintenance of genome integrity over cell divisions is critical for plant development and the correct transmission of genetic information to the progeny. A key factor involved in this process is the STRUCTURAL MAINTENANCE OF CHROMOSOME5 (SMC5) and SMC6 (SMC5/6) complex, related to the cohesin and condensin complexes that control sister chromatid alignment and chromosome condensation, respectively. Here, we characterize NON-SMC ELEMENT4 (NSE4) paralogs of the SMC5/6 complex in Arabidopsis (Arabidopsis thaliana). NSE4A is expressed in meristems and accumulates during DNA damage repair. Partial loss-of-function nse4a mutants are viable but hypersensitive to DNA damage induced by zebularine. In addition, nse4a mutants produce abnormal seeds, with noncellularized endosperm and embryos that maximally develop to the heart or torpedo stage. This phenotype resembles the defects in cohesin and condensin mutants and suggests a role for all three SMC complexes in differentiation during seed development. By contrast, NSE4B is expressed in only a few cell types, and loss-of-function mutants do not have any obvious abnormal phenotype. In summary, our study shows that the NSE4A subunit of the SMC5-SMC6 complex is essential for DNA damage repair in somatic tissues and plays a role in plant reproduction.

Boron (B), an essential micronutrient, causes adverse effects on the growth and development of plants when highly accumulated. By the analysis of Arabidopsis mutants hypersensitive to high-boron (high-B) stress, we have shown that 26S proteasome (26SP) is required to maintain the morphology of the root apical meristem (RAM) under high-B stress. To further understand the molecular function of 26SP in tolerance to high-B stress in the RAM, in this study we investigated the pathways regulated by 26SP using a 26SP subunit mutant, rpt5a, which is hypersensitive to high-B stress. Expression of RPT5a was induced by high-B stress in the entire RAM accompanied by its strong expression in the stele, including the stem cells. Analysis of stele organization in the rpt5a mutant revealed that 26SP is especially important for maintenance of the stele under high-B stress condition (3 mM B treatment). Expression analyses of an auxin-response reporter revealed that auxin responses were enhanced in the stele and the stem cell niche by high-B stress, especially in the rpt5a mutant. In contrast, the expression of TCS::GFP representing cytokinin signaling in the stem cell niche was unchanged in the wild type and extremely weak in the rpt5a mutant, irrespective of B condition. The drastically aberrant auxin and cytokinin responses in the rpt5a mutant under high-B stress were supported by transcriptome analysis using root tips. These results suggest that the collapse of hormonal crosstalk in the stele including the stem cells occurred in the rpt5a mutant, especially under high-B stress. Treatment with the auxin signaling inhibitor α-(phenyl ethyl-2-one)-indole-3-acetic acid (PEO-IAA) reduced sensitivity to high-B stress in the wild type and restored the RAM morphology in the rpt5a mutant under the high-B stress condition. In addition, cytokinin treatment conferred the rpt5a mutant with tolerance to high-B stress in RAM morphology. It is concluded that 26SP containing RPT5a is required for maintenance of auxin/cytokinin balance in the stele, which is crucial for preventing defects in RAM morphology under high-B stress.

Aurora kinase (AUR) is a well-known mitotic serine/threonine kinase that regulates centromere formation, chromosome segregation, and cytokinesis in eukaryotes. In addition to regulating mitotic events, AUR has been shown to regulate protein dynamics during interphase in animal cells. In contrast, there has been no identification and characterization of substrates and/or interacting proteins during interphase in plants. The Arabidopsis thaliana genome encodes three AUR paralogues, AtAUR1, AtAUR2, and AtAUR3. Among them, AtAUR1 and AtAUR2 are considered to function redundantly. Here, we confirmed that both AtAUR1 and AtAUR3 are localized in the nucleus and cytoplasm during interphase, suggesting that they have functions during interphase. To identify novel interacting proteins, we used AlphaScreen to target 580 transcription factors (TFs) that are mainly functional during interphase, using recombinant A. thaliana TFs and AtAUR1 or AtAUR3. We found 133 and 32 TFs had high potential for interaction with AtAUR1 and AtAUR3, respectively. The highly AtAUR-interacting TFs were involved in various biological processes, suggesting the functions of the AtAURs during interphase. We found that AtAUR1 and AtAUR3 showed similar interaction affinity to almost all TFs. However, in some cases, the interaction affinity differed substantially between the two AtAUR homologues. These results suggest that AtAUR1 and AtAUR3 have both redundant and distinct functions through interactions with TFs. In addition, database analysis revealed that most of the highly AtAUR-interacting TFs contained a detectable phosphopeptide that was consistent with the consensus motifs for human AURs, suggesting that these TFs are substrates of the AtAURs. The AtAURs phosphorylated several highly interacting TFs in the AlphaScreen in vitro. Overall, in line with the regulation of TFs through interaction, our results indicate the possibility of phosphoregulation of several TFs by the AtAURs (280/300).

Live imaging of the dynamics of nuclear organization provides the opportunity to uncover the mechanisms responsible for four-dimensional genome architecture. Here, we describe the use of fluorescent protein (FP) fusions of transcription activator-like effectors (TALEs) to visualize endogenous genomic sequences in Arabidopsis thaliana. The ability to engineer sequence-specific TALEs permits the investigation of precise genomic sequences. We could detect TALE-FP signals associated with centromeric, telomeric, and rDNA repeats and the signal distribution was consistent with that observed by fluorescent in situ hybridization. TALE-FPs are advantageous because they permit the observation of intact tissues. We used our TALE-FP method to investigate the nuclei of several multicellular plant tissues including roots, hypocotyls, leaves, and flowers. Because TALE-FPs permit live-cell imaging, we successfully observed the temporal dynamics of centromeres and telomeres in plant organs. Fusing TALEs to multimeric FPs enhanced the signal intensity when observing telomeres. We found that the mobility of telomeres was different in sub-nuclear regions. Transgenic plants stably expressing TALE-FPs will provide new insights into chromatin organization and dynamics in multicellular organisms., 植物のDNAを生きたまま観察できる手法を開発 --ゲノム編集のハサミをランプに替えることで作物のゲノム育種に貢献--. 京都大学プレスリリース. 2016-10-13.

Highlight Arabidopsis thaliana TPR5 is involved in proper alignment of the cell division plane and root elongation and is required for preventing micronuclei formation., Root growth in plants is achieved through the co-ordination of cell division and expansion. In higher plants, the radial structure of the roots is formed during embryogenesis and maintained thereafter throughout development. Here we show that the tetratricopeptide repeat domain protein TPR5 is necessary for maintaining radial structure and growth rates in Arabidopsis thaliana roots. We isolated an A. thaliana mutant with reduced root growth and determined that TPR5 was the gene responsible for the phenotype. The root growth rate of the tpr5-1 mutant was reduced to ~60% of that in wild-type plants. The radial structure was disturbed by the occurrence of occasional extra periclinal cell divisions. While the number of meristematic cells was reduced in the tpr5 mutants, the cell length in the mature portion of the root did not differ from that of the wild type, suggesting that TPR5 is required for proper cell division but dispensable for cell elongation. Expression of the TPR5–GFP fusion protein driven by the TPR5 promoter displayed fluorescence in the cytoplasm of root meristems, but not in mature root regions. DNA staining revealed that frequencies of micronuclei were increased in root meristems of tpr5 mutants. From this study, we concluded that TPR5 is involved in preventing the formation of micronuclei and is necessary for both the activity and directionality of cell division in root meristems.

The nucleolus, where components of the ribosome are constructed, is known to play an important role in various stress responses in animals. However, little is known about the role of the plant nucleolus under environmental stresses such as heat and chilling stress. In this study, we analyzed nucleolus morphology by determining the distribution of newly synthesized rRNAs with an analog of uridine, 5-ethynyl uridine (EU). When EU was incorporated into the root of the Arabidopsis thaliana, EU signals were strongly localized in the nucleolus. The results of the short-term incorporation of EU implied that there is no compartmentation among the processes of transcription, processing, and construction of rRNAs. Nevertheless, under heat and chilling stress, EU was not incorporated into the center of the nucleolus. Morphological analyses using whole rRNA staining and differential interference contrast observations revealed speckled and round structures in the center of the nucleolus under heat and chilling stress, respectively.

Somatic embryogenesis is one of the best examples of the remarkable developmental plasticity of plants, in which committed somatic cells can dedifferentiate and acquire the ability to form an embryo and regenerate an entire plant. In Arabidopsis thaliana, the shoot apices of young seedlings have been reported as an alternative tissue source for somatic embryos (SEs) besides the widely studied zygotic embryos taken from siliques. Although SE induction from shoots demonstrates the plasticity of plants more clearly than the embryo-to-embryo induction system, the underlying developmental and molecular mechanisms involved are unknown. Here we characterized SE formation from shoot apex explants by establishing a system for time-lapse observation of explants during SE induction. We also established a method to distinguish SE-forming and non-SE-forming explants prior to anatomical SE formation, enabling us to identify distinct transcriptome profiles of these two explants at SE initiation. We show that embryonic fate commitment takes place at day 3 of SE induction and the SE arises directly, not through callus formation, from the base of leaf primordia just beside the shoot apical meristem (SAM), where auxin accumulates and shoot-root polarity is formed. The expression domain of a couple of key developmental genes for the SAM transiently expands at this stage. Our data demonstrate that SE-forming and non-SE-forming explants share mostly the same transcripts except for a limited number of embryonic genes and root genes that might trigger the SE-initiation program. Thus, SE-forming explants possess a mixed identity (SAM, root and embryo) at the time of SE specification.

Xylem vessels are important for water conduction in vascular plants. The VASCULAR-RELATED NAC-DOMAIN (VND) family proteins, master regulators of xylem vessel cell differentiation in Arabidopsis thaliana, can upregulate a set of genes required for xylem vessel cell differentiation, including those involved in secondary cell wall (SCW) formation and programmed cell death (PCD), however, it is not fully understood how VND activity levels influence these processes. Here, we examined the Arabidopsis VND7-VP16-GR line, in which VND7 activity is post-translationally activated by treatments with different concentrations of dexamethasone (DEX), a synthetic glucocorticoid. Our observations showed that 1 nM DEX induced weak SCW deposition, but not PCD, whereas 10 or 100 nM DEX induced both SCW deposition and PCD. The decreased chlorophyll contents and SCW deposition were apparent after 24 h of 100 nM DEX treatment, but became evident only after 48 h of 10 nM DEX treatment. Moreover, the lower DEX concentrations delayed the upregulation of VND7 downstream genes, and decreased their induction levels. They collectively suggest that the regulation of VND activity is important not only to initiate xylem vessel cell differentiation, but also regulate the quality of the xylem vessels through VND-activity-dependent upregulation of the PCD- and SCW-related genes.

Pyrenocine A, a phytotoxin, was found to exhibit cytotoxicity against cancer cells with an IC50 value of 2.6–12.9 μM. Live cell imaging analysis revealed that pyrenocine A arrested HeLa cells at the M phase with characteristic ring-shaped chromosomes. Furthermore, as a result of immunofluorescence staining analysis, we found that pyrenocine A resulted in the formation of monopolar spindles in HeLa cells. Monopolar spindles are known to be induced by inhibitors of the kinesin motor protein Eg5 such as monastrol and STLC. Monastrol and STLC induce monopolar spindle formation and M phase arrest via inhibition of the ATPase activity of Eg5. Interestingly, our data revealed that pyrenocine A had no effect on the ATPase activity of Eg5 in vitro, which suggested the compound induces a monopolar spindle by an unknown mechanism. Structure-activity relationship analysis indicates that the enone structure of pyrenocine A is likely to be important for its cytotoxicity. An alkyne-tagged analog of pyrenocine A was synthesized and suppressed proliferation of HeLa cells with an IC50 value of 2.3 μM. We concluded that pyrenocine A induced monopolar spindle formation by a novel mechanism other than direct inhibition of Eg5 motor activity, and the activity of pyrenocine A may suggest a new anticancer mechanism.

Chromatin accessibility is closely associated with chromatin functions such as gene expression, DNA replication, and maintenance of DNA integrity. However, the relationship between chromatin accessibility and plant hormone signaling has remained elusive. Here, based on the correlation between chromatin accessibility and DNA damage, we used the sensitivity to DNA double strand breaks (DSBs) as an indicator of chromatin accessibility and demonstrated that auxin regulates chromatin accessibility through the TIR1/AFBs signaling pathway in proliferative cells. Treatment of proliferating plant cells with an inhibitor of the TIR1/AFBs auxin signaling pathway, PEO-IAA, caused chromatin loosening, indicating that auxin signaling functions to decrease chromatin accessibility. In addition, a transcriptome analysis revealed that several histone H4 genes and a histone chaperone gene, FAS1, are positively regulated through the TIR1/AFBs signaling pathway, suggesting that auxin plays a role in promoting nucleosome assembly. Analysis of the fas1 mutant of Arabidopsis thaliana confirmed that FAS1 is required for the auxin-dependent decrease in chromatin accessibility. These results suggest that the positive regulation of chromatin-related genes mediated by the TIR1/AFBs auxin signaling pathway enhances nucleosome assembly, resulting in decreased chromatin accessibility in proliferative cells.

Coherent X-ray diffraction imaging (CXDI) is a lens-less technique for visualizing the structures of non-crystalline particles with the dimensions of submicrometer to micrometer at a resolution of several tens of nanometers. We conducted cryogenic CXDI experiments at 66 K to visualize the internal structures of frozen-hydrated chloroplasts of Cyanidioschyzon merolae using X-ray free electron laser (XFEL) as a coherent X-ray source. Chloroplast dispersed specimen disks at a number density of 7/(10×10 µm(2)) were flash-cooled with liquid ethane without staining, sectioning or chemical labeling. Chloroplasts are destroyed at atomic level immediately after the diffraction by XFEL pulses. Thus, diffraction patterns with a good signal-to-noise ratio from single chloroplasts were selected from many diffraction patterns collected through scanning specimen disks to provide fresh specimens into the irradiation area. The electron density maps of single chloroplasts projected along the direction of the incident X-ray beam were reconstructed by using the iterative phase-retrieval method and multivariate analyses. The electron density map at a resolution of 70 nm appeared as a C-shape. In addition, the fluorescence image of proteins stained with Flamingo™ dye also appeared as a C-shape as did the autofluorescence from Chl. The similar images suggest that the thylakoid membranes with an abundance of proteins distribute along the outer membranes of chloroplasts. To confirm the present results statistically, a number of projection structures must be accumulated through high-throughput data collection in the near future. Based on the results, we discuss the feasibility of XFEL-CXDI experiments in the structural analyses of cellular organelles.

Nature Plants 3, 17097 (2017); published 26 June 2017; corrected 17 July 2017. The Supplementary Information files originally published with this Letter were not the intended final versions. The Supplementary Information PDF and Supplementary Tables 3, 5 and 6 have now been updated to fix a number of cosmetic problems.

Parasitic plants share a common anatomical feature, the haustorium. Haustoria enable both infection and nutrient transfer, which often leads to growth penalties for host plants and yield reduction in crop species. Haustoria also reciprocally transfer substances, such as RNA and proteins, from parasite to host, but the biological relevance for such movement remains unknown. Here, we studied such interspecies transport by using the hemiparasitic plant Phtheirospermum japonicum during infection of Arabidopsis thaliana Tracer experiments revealed a rapid and efficient transfer of carboxyfluorescein diacetate (CFDA) from host to parasite upon formation of vascular connections. In addition, Phtheirospermum induced hypertrophy in host roots at the site of infection, a form of enhanced secondary growth that is commonly observed during various parasitic plant-host interactions. The plant hormone cytokinin is important for secondary growth, and we observed increases in cytokinin and its response during infection in both host and parasite. Phtheirospermum-induced host hypertrophy required cytokinin signaling genes (AHK3,4) but not cytokinin biosynthesis genes (IPT1,3,5,7) in the host. Furthermore, expression of a cytokinin-degrading enzyme in Phtheirospermum prevented host hypertrophy. Wild-type hosts with hypertrophy were smaller than ahk3,4 mutant hosts resistant to hypertrophy, suggesting hypertrophy improves the efficiency of parasitism. Taken together, these results demonstrate that the interspecies movement of a parasite-derived hormone modified both host root morphology and fitness. Several microbial and animal plant pathogens use cytokinins during infections, highlighting the central role of this growth hormone during the establishment of plant diseases and revealing a common strategy for parasite infections of plants.

Plant microtubules (MTs) are nucleated from the γ-tubulin-containing ring complex (γTuRC). In cortical MT arrays of interphase plant cells, γTuRC is preferentially recruited to the lattice of preexisting MTs, where it initiates MT nucleation in either a branch- or bundle-forming manner, or dissociates without mediating nucleation. In this study, we analyzed how γTuRCs influence MT nucleation and dynamics in cotyledon pavement cells of

Scientific Reports 6: Article number: 29657; published online: 15 July 2016; updated: 13 January 2017 In the original version of this Article, all instances of “sGFP” were incorrectly given as “EGFP”. This error has now been corrected in the PDF and HTML versions of the Article.

The nucleus of Cyanidioschyzon merolae has quite a limited set of components. The core and linker histones, H2A.Z, and a centromeric histone variant CenH3 are preserved, but H2A variants including H2A.X, H2A.M, and H2A.W are absent. Some proteins of the nuclear pore complex and nucleoli are also preserved, but almost all homologs of nuclear lamina and nuclear envelope proteins are lacking. The chloroplast of this species is divided by a ring, which is composed of two inner rings, the FtsZ ring and inner plastid-dividing (PD) ring, and two outer rings, the outer PD ring and dynamin ring. Identification of the PD ring components in C. merolae enabled us to elucidate the molecular mechanism behind the PD machinery. For example, a glycosyltransferase protein, plastid-dividing ring 1, acts in establishing the PD ring by forming ring-shaped polyglucan filaments. The division of mitochondria is performed by the mitochondrion-dividing (MD), FtsZ, and dynamin rings. Mda1 functions as a mediator for stably linking a dynamin. In addition, an FtsZ-associated protein, ZED, is involved in completing the inner rings of the MD machinery. Analyses of the mechanisms of mitochondrial and chloroplast division in C. merolae contribute to our understanding of the endosymbiotic process of double-membrane-bounded organelles.

Coherent X-ray diffraction imaging (CXDI) is a lensless imaging technique for visualizing the structures of noncrystalline particles at a resolution of several tens of nanometers. The targets are particles with dimensions in the sub-micrometer to micrometer range. We carried out CXDI experiments at 66 K to investigate the internal structures of a whole frozen-hydrated cell and a chloroplast of Cyanidioschyzon merolae and a cyanobacteria cell by using coherent X-ray light sources, such as synchrotron and X-ray free-electron laser facilities. Owing to the short wavelength of the X-rays used (0.225 nm), the absorption and multiple scattering of X-rays inside the specimens were negligible. Diffraction patterns from each specimen particle adsorbed onto a thin membrane were collected at resolutions better than 50 μm−1 in reciprocal space. Therefore, structures of specimen particles can be illustrated at a resolution higher than 200 nm in real space. The most probable electron density map was retrieved from each diffraction pattern. The internal structures of the specimens are described here, particularly the structural correlation of the chloroplast of C. merolae and the cyanobacteria cell. Based on the experimental results, we discuss the feasibility of CXDI in the structural analyses of biological cells and cellular organelles.

Plant microtubules (MTs) are nucleated from the γ-tubulin-containing ring complex (γTuRC). In cortical MT arrays of interphase plant cells, γTuRC is preferentially recruited to the lattice of preexisting MTs, where it initiates MT nucleation in either a branch- or bundle-forming manner, or dissociates without mediating nucleation. In this study, we analyzed how γTuRCs influence MT nucleation and dynamics in cotyledon pavement cells of Arabidopsis thaliana We found that γTuRC nucleated MTs at angles of ∼40° toward the plus-ends of existing MTs, or in predominantly antiparallel bundles. A small fraction of γTuRCs was motile and tracked MT ends. When γTuRCs decorated the depolymerizing MT end, they reduced the depolymerization rate. Non-nucleating γTuRCs associated with the MT lattice promoted MT regrowth after a depolymerization phase. These results suggest that γTuRCs not only nucleate MT growth but also regulate MT dynamics by stabilizing MT ends. On rare occasions, a non-MT-associated γTuRC was pushed in the direction of the MT minus-end, while nucleating a new MT, suggesting that the polymerizing plus-end is anchored to the plasma membrane.