A leaf-emanated signal orchestrates grain size and number in response to maternal resources.

In plants, variations in seed size and number are outcomes of different reproductive strategies. Both traits are often environmentally influenced, suggesting that a mechanism exists to coordinate these phenotypes in response to available maternal resources. Yet, how maternal resources are sensed and influence seed size and number is largely unknown. Here, we report a mechanism that senses maternal resources and coordinates grain size and number in the wild rice Oryza rufipogon, a wild progenitor of Asian cultivated rice. We showed that FT-like 9 (FTL9) regulates both grain size and number and that maternal photosynthetic assimilates induce FTL9 expression in leaves to act as a long-range signal that increases grain number and reduces size. Our findings highlight a strategy that benefits wild plants to survive in a fluctuating environment. In this strategy, when maternal resources are sufficient, wild plants increase their offspring number while preventing an increase in offspring size by the action of FTL9, which helps expand their habitats. In addition, we found that a loss-of-function allele (ftl9) is prevalent among wild and cultivated populations, offering a new scenario in the history of rice domestication.

Comparison of constitutive promoter activities and development of maize ubiquitin promoter- and Gateway-based binary vectors for rice.

In transgenic experiments, we often face fundamental requirements such as overexpressing a certain gene, developing organelle markers, testing promoter activities, introducing large genomic fragments, and combinations of them. To fulfill these multiple requirements in rice, we developed simple binary vectors with or without maize ubiquitin (UBQ) promoter, Gateway cassette and fluorescent proteins. First, we compared stabilities of cauliflower mosaic virus 35S and maize UBQ promoters for constitutive gene expression in transgenic rice. We show that the 35S promoter was frequently silenced after shoot regeneration, whereas maize UBQ promoter achieved stable expression in various young tissues. Binary vectors with Gateway cassettes under the control of the UBQ promoter allowed us to develop stable organelle markers for nuclei, microtubules and P-bodies in rice. The maize UBQ promoter can be easily replaced with any promoters of interest as exemplified by reporters of mitotic cells and provascular bundles. Finally, by introducing two genomic fluorescent reporters, we showed utilities of the Gateway cassette and two selection markers in large DNA fragment transfer and sequential transformations, respectively. Thus, these binary vectors provide useful choices of transgenic experiments in rice.

A hierarchical transcriptional network activates specific CDK inhibitors that regulate G2 to control cell size and number in Arabidopsis.

How cell size and number are determined during organ development remains a fundamental question in cell biology. Here, we identified a GRAS family transcription factor, called SCARECROW-LIKE28 (SCL28), with a critical role in determining cell size in Arabidopsis. SCL28 is part of a transcriptional regulatory network downstream of the central MYB3Rs that regulate G2 to M phase cell cycle transition. We show that SCL28 forms a dimer with the AP2-type transcription factor, AtSMOS1, which defines the specificity for promoter binding and directly activates transcription of a specific set of SIAMESE-RELATED (SMR) family genes, encoding plant-specific inhibitors of cyclin-dependent kinases and thus inhibiting cell cycle progression at G2 and promoting the onset of endoreplication. Through this dose-dependent regulation of SMR transcription, SCL28 quantitatively sets the balance between cell size and number without dramatically changing final organ size. We propose that this hierarchical transcriptional network constitutes a cell cycle regulatory mechanism that allows to adjust cell size and number to attain robust organ growth.

Collection, preservation and distribution of Oryza genetic resources by the National Bioresource Project RICE (NBRP-RICE).

Biological resources are the basic infrastructure of bioscience research. Rice (Oryza sativa L.) is a good experimental model for research in cereal crops and monocots and includes important genetic materials used in breeding. The availability of genetic materials, including mutants, is important for rice research. In addition, Oryza species are attractive to researchers for both finding useful genes for breeding and for understanding the mechanism of genome evolution that enables wild plants to adapt to their own habitats. NBRP-RICE contributes to rice research by promoting the usage of genetic materials, especially wild Oryza accessions and mutant lines. Our activity includes collection, preservation and distribution of those materials and the provision of basic information on them, such as morphological and physiological traits and genomic information. In this review paper, we introduce the activities of NBRP-RICE and our database, Oryzabase, which facilitates the access to NBRP-RICE resources and their genomic sequences as well as the current situation of wild Oryza genome sequencing efforts by NBRP-RICE and other institutes.

High-resolution spatiotemporal transcriptome analyses during cellularization of rice endosperm unveil the earliest gene regulation critical for aleurone and starchy endosperm cell fate specification.

The major tissues of the cereal endosperm are the starchy endosperm (SE) in the inner and the aleurone layer (AL) at the outer periphery. The fates of the cells that comprise these tissues are determined according to positional information; however, our understanding of the underlying molecular mechanisms remains limited. Here, we conducted a high-resolution spatiotemporal analysis of the rice endosperm transcriptome during early cellularization. In rice, endosperm cellularization proceeds in a concentric pattern from a primary alveolus cell layer, such that developmental progression can be defined by the number of cell layers. Using laser-capture microdissection to obtain precise tissue sections, transcriptomic changes were followed through five histologically defined stages of cellularization from the syncytial to 3-cell layer (3 L) stage. In addition, transcriptomes were compared between the inner and the outermost peripheral cell layers. Large differences in the transcriptomes between stages and between the inner and the peripheral cells were found. SE attributes were expressed at the alveolus-cell-layer stage but were preferentially activated in the inner cell layers that resulted from periclinal division of the alveolus cell layer. Similarly, AL attributes started to be expressed only after the 2 L stage and were localized to the outermost peripheral cell layer. These results indicate that the first periclinal division of the alveolus cell layer is asymmetric at the transcriptome level, and that the cell-fate-specifying positional cues and their perception system are already operating before the first periclinal division. Several genes related to epidermal identity (i.e., type IV homeodomain-leucine zipper genes and wax biosynthetic genes) were also found to be expressed at the syncytial stage, but their expression was localized to the outermost peripheral cell layer from the 2 L stage onward. We believe that our findings significantly enhance our knowledge of the mechanisms underlying cell fate specification in rice endosperm.

OryzaGenome2.1: Database of Diverse Genotypes in Wild Oryza Species.

BACKGROUND: OryzaGenome ( http://viewer.shigen.info/oryzagenome21detail/index.xhtml ), a feature within Oryzabase ( https://shigen.nig.ac.jp/rice/oryzabase/ ), is a genomic database for wild Oryza species that provides comparative and evolutionary genomics approaches for the rice research community. RESULTS: Here we release OryzaGenome2.1, the first major update of OryzaGenome. The main feature in this version is the inclusion of newly sequenced genotypes and their meta-information, giving a total of 217 accessions of 19 wild Oryza species (O. rufipogon, O. barthii, O. longistaminata, O. meridionalis, O. glumaepatula, O. punctata, O. minuta, O. officinalis, O. rhizomatis, O. eichingeri, O. latifolia, O. alta, O. grandiglumis, O. australiensis, O. brachyantha, O. granulata, O. meyeriana, O. ridleyi, and O. longiglumis). These 19 wild species belong to 9 genome types (AA, BB, CC, BBCC, CCDD, EE, FF, GG, and HHJJ), representing wide genomic diversity in the genus. Using the genotype information, we analyzed the genome diversity of Oryza species. Other features of OryzaGenome facilitate the use of information on single nucleotide polymorphisms (SNPs) between O. sativa and its wild progenitor O. rufipogon in rice research, including breeding as well as basic science. For example, we provide Variant Call Format (VCF) files for genome-wide SNPs of 33 O. rufipogon accessions against the O. sativa reference genome, IRGSP1.0. In addition, we provide a new SNP Effect Table function, allowing users to identify SNPs or small insertion/deletion polymorphisms in the 33 O. rufipogon accessions and to search for the effect of these polymorphisms on protein function if they reside in the coding region (e.g., are missense or nonsense mutations). Furthermore, the SNP Viewer for 446 O. rufipogon accessions was updated by implementing new tracks for possible selective sweep regions and highly mutated regions that were potentially exposed to selective pressures during the process of domestication. CONCLUSION: OryzaGenome2.1 focuses on comparative genomic analysis of diverse wild Oryza accessions collected around the world and on the development of resources to speed up the identification of critical trait-related genes, especially from O. rufipogon. It aims to promote the use of genotype information from wild accessions in rice breeding and potential future crop improvements. Diverse genotypes will be a key resource for evolutionary studies in Oryza, including polyploid biology.

Agrobacterium-Mediated Genetic Transformation of Wild Oryza Species Using Immature Embryos.

Genetic transformation is one of the most important technologies for revealing or modulating gene function. It is used widely in both functional genomics and molecular breeding of rice. Demands on its use in wild Oryza species is increasing because of their high genetic diversity. Given the difficulties in genetic crosses between distantly related species, genetic transformation offers a way to alter or transfer genetic traits in wild rice accessions. However, transformation of wild Oryza accessions by conventional methods using calli induced from scutellum tissue of embryos in mature seeds often fails. Here, we report methods using immature embryos for the genetic transformation of a broad range of Oryza species. First, we investigated the ability of callus induction and regeneration from immature embryos of 192 accessions in 20 species under several culture conditions. We regenerated plants from immature embryos of 90 accessions in 16 species. Next, we optimized the conditions of Agrobacterium infection using a vector carrying the GFP gene driven by the maize ubiquitin promoter. GFP signals were observed in 51 accessions in 11 species. We analyzed the growth and seed set of transgenic plants of O. barthii, O. glumaepatula, O. rufipogon, and O. brachyantha. The plants grew to maturity and set seeds normally. Southern blot analyses using DNA from T0 plants showed that all GFP plants were derived from independent transformation events. We confirmed that the T-DNAs were transmitted to the next generation through the segregation of GFP signals in the T1 generation. These results show that many Oryza species can be transformed by using modified immature-embryo methods. This will accelerate the use of wild Oryza accessions in molecular genetic analyses and molecular breeding.

Specification of basal region identity after asymmetric zygotic division requires mitogen-activated protein kinase 6 in rice.

Asymmetric cell division is a key step in cellular differentiation in multicellular organisms. In plants, asymmetric zygotic division produces the apical and basal cells. The mitogen-activated protein kinase (MPK) cascade in Arabidopsis acts in asymmetric divisions such as zygotic division and stomatal development, but whether the effect on cellular differentiation of this cascade is direct or indirect following asymmetric division is not clear. Here, we report the analysis of a rice mutant, globular embryo 4 (gle4). In two- and four-cell-stage embryos, asymmetric zygotic division and subsequent cell division patterns were indistinguishable between the wild type and gle4 mutants. However, marker gene expression and transcriptome analyses showed that specification of the basal region was compromised in gle4 We found that GLE4 encodes MPK6 and that GLE4/MPK6 is essential in cellular differentiation rather than in asymmetric zygotic division. Our findings provide a new insight into the role of MPK in plant development. We propose that the regulation of asymmetric zygotic division is separate from the regulation of cellular differentiation that leads to apical-basal polarity.

Development of the Mitsucal computer system to identify causal mutation with a high-throughput sequencer.

KEY MESSAGE: Development of Mitsucal. Recent advances in DNA sequencing technology have facilitated whole-genome sequencing of mutants and variants. However, the analyses of large sequence datasets using a computer remain more difficult than operating a sequencer. Forward genetic approach is powerful even in sexual reproduction to identify key genes. Therefore, we developed the Mitsucal computer system for identifying causal genes of mutants, using whole-genome sequence data. Mitsucal includes a user-friendly web interface to configure analysis variables, such as background and crossed strains. Other than configuration, users are only required to upload short reads. All results are presented through a web interface where users can easily obtain a short list of candidate mutations. In the present study, we present three examples of Arabidopsis mutants defective in sexual reproduction in which Mitsucal is used to identify causal mutation. One mutant was screened from seeds of a transgenic line with a reporter gene to elucidate the mechanisms involved in the regulation of seed oil storage. The identified gene codes for a protein may be involved in mRNA splicing. Other two mutants had defects in the surface walls on pollen termed exine. Both causal genes were identified, and mutants were found to be allele of known mutants. These results show that Mitsucal could facilitate identification of causal genes.

KNS4/UPEX1: A Type II Arabinogalactan ß-(1,3)-Galactosyltransferase Required for Pollen Exine Development.

Pollen exine is essential for protection from the environment of the male gametes of seed-producing plants, but its assembly and composition remain poorly understood. We previously characterized Arabidopsis (Arabidopsis thaliana) mutants with abnormal pollen exine structure and morphology that we named kaonashi (kns). Here we describe the identification of the causal gene of kns4 that was found to be a member of the CAZy glycosyltransferase 31 gene family, identical to UNEVEN PATTERN OF EXINE1, and the biochemical characterization of the encoded protein. The characteristic exine phenotype in the kns4 mutant is related to an abnormality of the primexine matrix laid on the surface of developing microspores. Using light microscopy with a combination of type II arabinogalactan (AG) antibodies and staining with the arabinogalactan-protein (AGP)-specific ß-Glc Yariv reagent, we show that the levels of AGPs in the kns4 microspore primexine are considerably diminished, and their location differs from that of wild type, as does the distribution of pectin labeling. Furthermore, kns4 mutants exhibit reduced fertility as indicated by shorter fruit lengths and lower seed set compared to the wild type, confirming that KNS4 is critical for pollen viability and development. KNS4 was heterologously expressed in Nicotiana benthamiana, and was shown to possess ß-(1,3)-galactosyltransferase activity responsible for the synthesis of AG glycans that are present on both AGPs and/or the pectic polysaccharide rhamnogalacturonan I. These data demonstrate that defects in AGP/pectic glycans, caused by disruption of KNS4 function, impact pollen development and viability in Arabidopsis.

MYB3Rs, plant homologs of Myb oncoproteins, control cell cycle-regulated transcription and form DREAM-like complexes.

Plant MYB3R transcription factors, homologous to Myb oncoproteins, regulate the genes expressed at G2 and M phases in the cell cycle. Recent studies showed that MYB3Rs constitute multiprotein complexes that may correspond to animal complexes known as DREAM or dREAM. Discovery of the putative homologous complex in plants uncovered their significant varieties in structure, function, dynamics, and heterogeneity, providing insight into conserved and diversified aspects of cell cycle-regulated gene transcription.

Transcriptional repression by MYB3R proteins regulates plant organ growth.

In multicellular organisms, temporal and spatial regulation of cell proliferation is central for generating organs with defined sizes and morphologies. For establishing and maintaining the post-mitotic quiescent state during cell differentiation, it is important to repress genes with mitotic functions. We found that three of the Arabidopsis MYB3R transcription factors synergistically maintain G2/M-specific genes repressed in post-mitotic cells and restrict the time window of mitotic gene expression in proliferating cells. The combined mutants of the three repressor-type MYB3R genes displayed long roots, enlarged leaves, embryos, and seeds. Genome-wide chromatin immunoprecipitation revealed that MYB3R3 binds to the promoters of G2/M-specific genes and to E2F target genes. MYB3R3 associates with the repressor-type E2F, E2FC, and the RETINOBLASTOMA RELATED proteins. In contrast, the activator MYB3R4 was in complex with E2FB in proliferating cells. With mass spectrometry and pairwise interaction assays, we identified some of the other conserved components of the multiprotein complexes, known as DREAM/dREAM in human and flies. In plants, these repressor complexes are important for periodic expression during cell cycle and to establish a post-mitotic quiescent state determining organ size.

Genetic interaction between G2/M phase-specific transcription factor MYB3R4 and MAPKKK ANP3 for execution of cytokinesis in Arabidopsis thaliana.

Plant cells are surrounded by rigid cell walls, and hence, their division is associated with a plant-specific mode of cytokinesis in which the cell plate, a new cell wall, is generated and separates 2 daughter nuclei. The successful execution of cytokinesis requires the timely activation of multiple regulatory pathways, which include the AtNACK1/HINKEL kinesin-induced MAPK cascade and MYB3R1/4-mediated transcriptional activation of G2/M-specific genes. However, it remains unclear whether and how these pathways are functionally interconnected to each other. By analyzing enhancer mutations of myb3r4, here we found a close genetic interaction between the 2 pathways; a mutation in ANP3, which encodes MAPKKK (acting downstream of AtNACK1/HINKEL), strongly enhanced the defective cytokinesis observed in the myb3r4 mutant. This interaction may not be due to the direct activation of MYB3R1/4 by the MAPK cascade; rather, possibly to the downstream targets of these 2 signaling pathways, acting in close proximity. Our results showed that MYB3R1/4 may positively affect cytokinesis via multiple pathways, one of which may act independently from the KNOLLE-dependent pathway defined previously, and affect the downstream events that may also be under the control of the AtNACK1/HINKEL-mediated MAPK cascade.

Self-disproportionation of enantiomers of thalidomide and its fluorinated analogue via gravity-driven achiral chromatography: mechanistic rationale and implications.

We report on the self-disproportionation of enantiomers (SDE) of non-racemic thalidomide (1) and 3'-fluorothalidomide (2) under the conditions of gravity-driven achiral silica-gel chromatography. The presence of a fluorine atom on the chiral center dramatically alters the structure and polarity of 1 and 2, resulting in the opposite SDE profile on silica-gel.

Development and disintegration of tapetum-specific lipid-accumulating organelles, elaioplasts and tapetosomes, in Arabidopsis thaliana and Brassica napus.

The pollen coat covering the surface of pollen grains has many important roles for pollination. In Brassicaceae plants, the pollen coat components are synthesized and temporarily accumulated in two tapetum-specific organelles, the elaioplast and the tapetosome. Although many biochemical and electron microscopic analyses have been attempted, the structure and biogenesis of these organelles have not been fully elucidated. To resolve this problem, we performed live imaging of these organelles using two markers, FIB1a-GFP and GRP17-GFP. FIB1a is an Arabidopsis fibrillin, a structural protein of elaioplast plastoglobules. In transgenic Arabidopsis, fluorescence of FIB1a-GFP appeared in young elaioplasts, in which small plastoglobules were developing. However, the fluorescence disappeared in later stages, while enlargement of plastoglobules continued. GRP17 is an Arabidopsis oleopollenin, an oleosin-like protein in tapetosomes. Fluorescence microscopy of GRP17-GFP expressed in Arabidopsis and Brassica napus revealed that tapetosomes do not contain oleopollenin-coated vesicles but have an outer envelope, indicating that the tapetosome structure is distinct from seed oil bodies. Visualization of GRP17-GFP also demonstrated that the tapetal cells become protoplasts and migrate into locules before pollen coat formation, and provided live imaging of the foot formation between pollen grains and stigmatic papilla cells.

Cosuppression of NtmybA1 and NtmybA2 causes downregulation of G2/M phaseexpressed genes and negatively affects both cell division and expansion in tobacco.

During the plant cell cycle, genes preferentially expressed at the G2/M phase are regulated by R1R2R3-type Myb transcription factors. To address the function of 2 tobacco R1R2R3-Myb proteins, NtmybA1 and NtmybA2, we generated transgenic tobacco plants in which endogenous NtmybA2 transcripts were significantly decreased, presumably due to cosuppression triggered by the presence of the NtmybA2 transgene. These lines showed a concomitant downregulation of structurally related NtmybA1 and many G2/M-expressed genes. In the cosuppression plants, we found a dwarf phenotype due to both reduced cell size and decreased cell number. Our results provide evidence confirming our previous view that NtmybA1 and NtmybA2 may regulate cell expansion as well as cell division by transcriptionally activating many G2/M-expressed genes in tobacco.

Acute heart failure syndrome associated with snow shoveling.

There have been only a few reports regarding the relation between snow shoveling and acute heart failure syndromes (AHFS). We present a case series of 5 patients who presented with AHFS, all within 5 days after shoveling snow. Although all patients underwent examination at a regular out-patient clinic, no patient had prior signs or symptoms of heart failure. The condition of all patients had gradually deteriorated, with no abrupt onset of dyspnea after shoveling snow. Four of the 5 patients demonstrated a preserved ejection fraction on echocardiography. Snow shoveling may lead to AHFS in patients who are at risk for developing heart failure.

Absolute chirality of the γ-polymorph of glycine: correlation of the absolute structure with the optical rotation.

Crystal specimens of the γ-polymorph of achiral glycine which crystallize in space groups P31 and P32 as determined by the anomalous X-ray scattering method are shown to be laevorotatory and dextrorotatory, respectively, as determined by optical rotation of the crystals.

The present state and problems of graduate-entry programs (GEP) in national medical schools in Japan.

It is not certain whether graduate-entry program (GEP) or non-graduate-entry program (non-GEP) in medical education is desirable to foster excellent medical physicians in Japan. In order to clarify the present state and problems of GEP, we visited 27 national medical schools which have introduced limited term college graduate-entry program and discussed with the deans, the education committee and administration affairs.GEP students are elder and usually study harder than regular non-GEP course students. Therefore, they got the higher grades at 1-2 classes of GEP course than the regular non-GEP course students. However, some GEP students lost motivation to study medicine and got poor grades at higher class. There was no definite difference of the final grades at the graduation between GEP and non-GEP students. Most of GEP students became medical practitioners and few students chose physician scientist majoring in basic medicine.We did not find any advantage of GEP compared with regular non-GEP. The results show that the introduction of GEP throughout Japan should be discussed carefully.

The current medical education system in the world.

To contribute to the innovation of the medical education system in Japan, we visited 35 medical schools and 5 institutes in 12 countries of North America, Europe, Australia and Asia in 2008-2010 and observed the education system. We met the deans, medical education committee and administration affairs and discussed about the desirable education system. We also observed the facilities of medical schools.Medical education system shows marked diversity in the world. There are three types of education course; non-graduate-entry program(non-GEP), graduate-entry program(GEP) and mixed program of non-GEP and GEP. Even in the same country, several types of medical schools coexist. Although the education methods are also various among medical schools, most of the medical schools have introduced tutorial system based on PBL or TBL and simulation-based learning to create excellent medical physicians. The medical education system is variable among countries depending on the social environment. Although the change in education program may not be necessary in Japan, we have to innovate education methods; clinical training by clinical clerkship must be made more developed to foster the training of the excellent clinical physicians, and tutorial education by PBL or TBL and simulation-based learning should be introduced more actively.