Evolutionary alterations in gene expression and enzymatic activities of gibberellin 3-oxidase 1 in Oryza.

Proper anther and pollen development are important for plant reproduction. The plant hormone gibberellin is important for anther development in rice, but its gametophytic functions remain largely unknown. Here, we report the functional and evolutionary analyses of rice gibberellin 3-oxidase 1 (OsGA3ox1), a gibberellin synthetic enzyme specifically expressed in the late developmental stages of anthers. Enzymatic and X-ray crystallography analyses reveal that OsGA3ox1 has a higher GA7 synthesis ratio than OsGA3ox2. In addition, we generate an osga3ox1 knockout mutant by genome editing and demonstrate the bioactive gibberellic acid synthesis by the OsGA3ox1 action during starch accumulation in pollen via invertase regulation. Furthermore, we analyze the evolution of Oryza GA3ox1s and reveal that their enzyme activity and gene expression have evolved in a way that is characteristic of the Oryza genus and contribute to their male reproduction ability.

Mutation of the imprinted gene OsEMF2a induces autonomous endosperm development and delayed cellularization in rice.

In angiosperms, endosperm development comprises a series of developmental transitions controlled by genetic and epigenetic mechanisms that are initiated after double fertilization. Polycomb repressive complex 2 (PRC2) is a key component of these mechanisms that mediate histone H3 lysine 27 trimethylation (H3K27me3); the action of PRC2 is well described in Arabidopsis thaliana but remains uncertain in cereals. In this study, we demonstrate that mutation of the rice (Oryza sativa) gene EMBRYONIC FLOWER2a (OsEMF2a), encoding a zinc-finger containing component of PRC2, causes an autonomous endosperm phenotype involving proliferation of the central cell nuclei with separate cytoplasmic domains, even in the absence of fertilization. Detailed cytological and transcriptomic analyses revealed that the autonomous endosperm can produce storage compounds, starch granules, and protein bodies specific to the endosperm. These events have not been reported in Arabidopsis. After fertilization, we observed an abnormally delayed developmental transition in the endosperm. Transcriptome and H3K27me3 ChIP-seq analyses using endosperm from the emf2a mutant identified downstream targets of PRC2. These included >100 transcription factor genes such as type-I MADS-box genes, which are likely required for endosperm development. Our results demonstrate that OsEMF2a-containing PRC2 controls endosperm developmental programs before and after fertilization.

Evolution and Diversity of the Wild Rice Oryza officinalis Complex, across Continents, Genome Types, and Ploidy Levels.

The Oryza officinalis complex is the largest species group in Oryza, with more than nine species from four continents, and is a tertiary gene pool that can be exploited in breeding programs for the improvement of cultivated rice. Most diploid and tetraploid members of this group have a C genome. Using a new reference C genome for the diploid species O. officinalis, and draft genomes for two other C genome diploid species Oryza eichingeri and Oryza rhizomatis, we examine the influence of transposable elements on genome structure and provide a detailed phylogeny and evolutionary history of the Oryza C genomes. The O. officinalis genome is 1.6 times larger than the A genome of cultivated Oryza sativa, mostly due to proliferation of Gypsy type long-terminal repeat transposable elements, but overall syntenic relationships are maintained with other Oryza genomes (A, B, and F). Draft genome assemblies of the two other C genome diploid species, Oryza eichingeri and Oryza rhizomatis, and short-read resequencing of a series of other C genome species and accessions reveal that after the divergence of the C genome progenitor, there was still a substantial degree of variation within the C genome species through proliferation and loss of both DNA and long-terminal repeat transposable elements. We provide a detailed phylogeny and evolutionary history of the Oryza C genomes and a genomic resource for the exploitation of the Oryza tertiary gene pool.

Overcoming the species hybridization barrier by ploidy manipulation in the genus Oryza.

In most eudicot and monocot species, interspecific and interploidy crosses generally display abnormalities in the endosperm that are the major cause of a post-zygotic hybridization barrier. In some eudicot species, however, this type of hybridization barrier can be overcome by the manipulation of ploidy levels of one parental species, suggesting that the molecular mechanisms underlying the species hybridization barrier can be circumvented by genome dosage. We previously demonstrated that endosperm barriers in interspecific and interploidy crosses in the genus Oryza involve overlapping but different mechanisms. This result contrasts with those in the genus Arabidopsis, which shows similar outcomes in both interploidy and interspecific crosses. Therefore, we postulated that an exploration of pathways for overcoming the species hybridization barrier in Oryza endosperm, by manipulating the ploidy levels in one parental species, might provide novel insights into molecular mechanisms. We showed that fertile hybrid seeds could be produced by an interspecific cross of female tetraploid Oryza sativa and male diploid Oryza longistaminata. Although the rate of nuclear divisions did not return to normal levels in the hybrid endosperm, the timing of cellularization, nucellus degeneration and the accumulation of storage products were close to normal levels. In addition, the expression patterns of the imprinted gene MADS87 and YUCCA11 were changed when the species barrier was overcome. These results suggest that the regulatory machinery for developmental transitions and imprinted gene expression are likely to play a central role in overcoming species hybridization barriers by genome dosage in the genus Oryza.

Dissection of two major components of the post-zygotic hybridization barrier in rice endosperm.

A post-zygotic hybridization barrier is often observed in the endosperm of seeds produced by interspecific or interploidy crosses. In Arabidopsis thaliana, for example, hybrid endosperm from both types of cross shows altered timing of cellularization and an altered rate of nuclear divisions. Therefore, it has been proposed that interspecific and interploidy crosses share common molecular mechanisms for establishment of an effective species barrier. However, these two types of hybridization barrier may be initiated by different intrinsic cues: the interspecific cross barrier arises after hybridization of genomes with differences in DNA sequences, while the interploidy cross barrier arises after hybridization of genomes with the same DNA sequences but differences in ploidy levels. In this study, we performed interploidy crosses to identify components of the post-hybridization barrier in the endosperm of rice. We performed an intra-cultivar cross of autotetraploid (4n) × diploid (2n) rice, and found precocious cellularization and a decreased rate of nuclear division in the syncytial endosperm. By contrast, seeds from the reciprocal cross showed delayed cellularization and an increased rate of nuclear division. This differential effect on nuclear division rates contrasts with the outcome of rice interspecific crosses, which were previously shown to have altered timing of cellularization without any change in nuclear division rates. Thus, we propose that the post-zygotic hybridization barrier in rice endosperm has two separable components, namely control of the timing of cellularization and control of the nuclear division rates in the syncytial stage of endosperm development.

A map of rice genome variation reveals the origin of cultivated rice.

Crop domestications are long-term selection experiments that have greatly advanced human civilization. The domestication of cultivated rice (Oryza sativa L.) ranks as one of the most important developments in history. However, its origins and domestication processes are controversial and have long been debated. Here we generate genome sequences from 446 geographically diverse accessions of the wild rice species Oryza rufipogon, the immediate ancestral progenitor of cultivated rice, and from 1,083 cultivated indica and japonica varieties to construct a comprehensive map of rice genome variation. In the search for signatures of selection, we identify 55 selective sweeps that have occurred during domestication. In-depth analyses of the domestication sweeps and genome-wide patterns reveal that Oryza sativa japonica rice was first domesticated from a specific population of O. rufipogon around the middle area of the Pearl River in southern China, and that Oryza sativa indica rice was subsequently developed from crosses between japonica rice and local wild rice as the initial cultivars spread into South East and South Asia. The domestication-associated traits are analysed through high-resolution genetic mapping. This study provides an important resource for rice breeding and an effective genomics approach for crop domestication research.

ICF syndrome in a girl with DNA hypomethylation but without detectable DNMT3B mutation.

A 3-year-old girl with phenotypic and cytogenetic manifestations of the ICF syndrome and DNA hypomethylation but without DNMT3B gene mutation is described. At age 3 months, she had an apneic spell that left her with spastic paraplegia and severe mental retardation. At age 8 months, she suffered meningococcal meningitis and sepsis. When seen by us at age 3 years with virilization, she had a cleft plate, macroglossia, and an atrial septal defect. An adenoma was surgically removed from the right adrenal cortex. Her serum immunoglobulin levels were normal except IgA at the low normal border. Her lymphocytes showed paracentromeric stretching of chromosomes 1 and 16 in 7% of metaphases, and multiradial figures involving these chromosomes in 1% of cells. Hypomethylation of classical satellite 2 DNA was observed with BstBI digestion, but in a lesser degree than those in the individuals with proven DNMT3B mutations. No mutation was found in the coding and promoter regions of the gene. Several alternative interpretations were considered to explain the low frequencies of chromosomal instabilities and the lower degree of DNA hypomethylation, and undetected DNA3B mutations. A mutation may be present in the gene but undetected, present in other DNA methyltransferases (DNMT) genes or in a DNMT-associated protein gene.

Disruption of mesodermal enhancers for Igf2 in the minute mutant.

The radiation-induced mutation minute (Mnt) in the mouse leads to intrauterine growth retardation with paternal transmission and has been linked to the distal chromosome 7 cluster of imprinted genes. We show that the mutation is an inversion, whose breakpoint distal to H19 disrupts and thus identifies an enhancer for Igf2 expression in skeletal muscle and tongue, and separates the gene from other mesodermal and extra-embryonic enhancers. Paternal transmission of Mnt leads to drastic downregulation of Igf2 transcripts in all mesodermal tissues and the placenta. Maternal transmission leads to methylation of the H19 differentially methylated region (DMR) and silencing of H19, showing that elements 3' of H19 can modify the maternal imprint. Methylation of the maternal DMR leads to biallelic expression of Igf2 in endodermal tissues and foetal overgrowth, demonstrating that methylation in vivo can open the chromatin boundary upstream of H19. Our work shows that most known enhancers for Igf2 are located 3' of H19 and establishes an important genetic paradigm for the inheritance of complex regulatory mutations in imprinted gene clusters.