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.

Control of Plant Cell Growth and Proliferation by MO25A, a Conserved Major Component of the Mammalian Sterile 20-Like Kinase Pathway.

The precise control of cell growth and proliferation underpins the development of plants and animals. These factors affect the development and size of organs and the body. In plants, the growth and proliferation of cells are regulated by environmental stimuli and intrinsic signaling, allowing different cell types to have specific growth and proliferation characteristics. An increasing number of factors that control cell division and growth have been identified. However, the mechanisms underlying cell type-specific cell growth and proliferation characteristics in the normal developmental context are poorly understood. Here, we analyzed the rice mutant osmo25a1, which is defective in the progression of embryogenesis. The osmo25a1 mutant embryo developed incomplete embryonic organs, such as the shoot and root apical meristems. It showed a delayed progression of embryogenesis, associated with the reduced mitotic activity. The causal gene of this mutation encodes a member of the Mouse protein-25A (MO25A) family of proteins that have pivotal functions in a signaling pathway that governs cell proliferation and polarity in animals, yeasts and filamentous fungi. To elucidate the function of plant MO25A at the cellular level, we performed a functional analysis of MO25A in the moss Physcomitrium patens. Physcomitrium patens MO25A was uniformly distributed in the cytoplasm and functioned in cell tip growth and the initiation of cell division in stem cells. Overall, we demonstrated that MO25A proteins are conserved factors that control cell proliferation and growth.

Post-embryonic function of GLOBULAR EMBRYO 4 (GLE4)/OsMPK6 in rice development.

In plants, mitogen activated protein kinases (MPKs) are involved in various signaling pathways that lead to biotic and abiotic responses as well as that regulate developmental processes. Among them, MPK6 and its closely related homologue, MPK3, act redundantly and are known to be involved in asymmetric cell divisions of meristemoid mother cells in stomata development and of zygotes in Arabidopsis. Loss-of-function mutants of GLE4/OsMPK6, which is an orthologue of MPK6 in rice, showed a defect in polarity establishment in early stage of embryogenesis. However, because of the embryo lethality of the mutations, the function of GLE4/OsMPK6 in post-embryonic development is not clarified. Here, we report the analysis of post embryonic function of GLE4/OsMPK6 in vegetative stage of rice using regenerated gle4/osmpk6 homozygous plants from tissue culture. The regenerated plants are dwarf and produce multiple shoots with small leaves. These shoots never develop into reproductive stage, instead, proliferate vegetative shoots repeatedly. Leaves of gle4/osmpk6 have small leaf blade at the tip and blade-sheath boundary become obscure. Stomata arrangement is also disturbed in gle4/osmpk6 leaf blade. The shape of shoot apical meristem of gle4/osmpk6 become disorganized. Thus, GLE4/OsMPK6 functions in shoot organization and stomata patterning in the post embryonic development in rice.

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.

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.

Genome-wide analysis of spatiotemporal gene expression patterns during early embryogenesis in rice.

Embryogenesis in rice is different from that of most dicotolydonous plants in that it shows a non-stereotypic cell division pattern, formation of dorsal-ventral polarity, and endogenous initiation of the radicle. To reveal the transcriptional features associated with developmental events during rice early embryogenesis, we used microarray analysis coupled with laser microdissection to obtain both spatial and temporal transcription profiles. Our results allowed us to determine spatial expression foci for each expressed gene in the globular embryo, which revealed the importance of phytohormone-related genes and a suite of transcription factors to early embryogenesis. Our analysis showed the polarized expression of a small number of genes along the apical-basal and dorsal-ventral axes in the globular embryo, which tended to fluctuate in later developmental stages. We also analyzed gene expression patterns in the early globular embryo and how this relates to expression in embryonic organs at later stages. We confirmed the accuracy of the expression patterns found by microarray analysis of embryo subdomains using in situ hybridization. Our study identified homologous genes from Arabidopsis thaliana with known functions in embryogenesis in addition to unique and uncharacterized genes that show polarized expression patterns during embryogenesis. The results of this study are presented in a database to provide a framework for spatiotemporal gene expression during rice embryogenesis, to serve as a resource for future functional analysis of genes, and as a basis for comparative studies of plant embryogenesis.

Expression of the rice microRNA miR820 is associated with epigenetic modifications at its own locus.

Small RNAs, such as small interfering RNAs (siRNAs) or microRNAs (miRNAs), regulate gene expression at transcriptional and posttranscriptional levels in eukaryotes. miRNAs are processed from duplexes formed on single-stranded RNA. They regulate expression of their target gene either by cleaving mRNA or supressing translation. In general, the primary miRNA transcripts are synthesized by RNA polymerase II and processed similarly to mRNAs. MIRNA genes are usually located in transcriptionally active euchromatic regions. In contrast, siRNAs are processed from duplexes made of two RNA molecules. One of them is often derived from a transposable element (TE) or from repetitive sequences that reside in heterochromatic regions. The other strand is synthesized by the RNA-dependent RNA polymerase on the first strand as a template. siRNAs establish epigenetic marks in parasitic DNA such as TEs, thus they usually act in cis. The rice miRNA miR820, encoded by CACTA TEs (five copies, located on different chromosomes), reduces the expression of the de novo DNA methyltransferase gene OsDRM2. Because miR820 is derived from silent TEs, in which the heterochromatic histone modifications are enriched, the mechanism of MIR820 transcription could be expected to differ from typical miRNAs. Here we show that the primary transcript of MIR820 is mainly derived from the CACTA TE copy on chromosome 7 (MIR820b). Histone modification and DNA methylation status around MIR820b differed from that of the other four loci. These unique epigenetic modifications in MIR820b were only found around the miR820 coding region. We conclude that MIR820b transcription may depend on the unique epigenetic modifications, which in turn may be established by the action of miR820 in cis. This suggests a dual function of miR820 in cis and in trans.

The copy number of rice CACTA DNA transposons carrying MIR820 does not correlate with MIR820 expression.

MiR820 is a small RNA species (22 and 24 nucleotides), produced from transcripts originated from a region inside CACTA DNA transposons in rice. Because MIR820 is a transposon gene, its expression may depend on the transposon copy number. Here, we investigated the copy number of MIR820 and its expression levels in various cultivars and wild species of rice. We found no correlation between copy number and expression level, suggesting that MIR820 transcription is regulated not by the copy dosage but by the epigenetic state of each copy.

Two WUSCHEL-related homeobox genes, narrow leaf2 and narrow leaf3, control leaf width in rice.

Leaf shape is one of the key determinants of plant architecture. Leaf shape also affects the amount of sunlight captured and influences photosynthetic efficiency; thus, it is an important agronomic trait in crop plants. Understanding the molecular mechanisms governing leaf shape is a central issue of plant developmental biology and agrobiotechnology. Here, we characterized the narrow-leaf phenotype of FL90, a linkage tester line of rice (Oryza sativa). Light and scanning electron microscopic analyses of FL90 leaves revealed defects in the development of marginal regions and a reduction in the number of longitudinal veins. The narrow-leaf phenotype of FL90 shows a two-factor recessive inheritance and is caused by the loss of function of two WUSCHEL-related homeobox genes, NAL2 and NAL3 (NAL2/3), which are duplicate genes orthologous to maize NS1 and NS2 and to Arabidopsis PRS. The overexpression of NAL2/3 in transgenic rice plants results in wider leaves containing increased numbers of veins, suggesting that NAL2/3 expression regulates leaf width. Thus, NAL2/3 can be used to modulate leaf shape and improve agronomic yield in crop plants.

LAX PANICLE2 of rice encodes a novel nuclear protein and regulates the formation of axillary meristems.

Aerial architecture in higher plants is dependent on the activity of the shoot apical meristem (SAM) and axillary meristems (AMs). The SAM produces a main shoot and leaf primordia, while AMs are generated at the axils of leaf primordia and give rise to branches and flowers. Therefore, the formation of AMs is a critical step in the construction of plant architecture. Here, we characterized the rice (Oryza sativa) lax panicle2 (lax2) mutant, which has altered AM formation. LAX2 regulates the branching of the aboveground parts of a rice plant throughout plant development, except for the primary branch in the panicle. The lax2 mutant is similar to lax panicle1 (lax1) in that it lacks an AM in most of the lateral branching of the panicle and has a reduced number of AMs at the vegetative stage. The lax1 lax2 double mutant synergistically enhances the reduced-branching phenotype, indicating the presence of multiple pathways for branching. LAX2 encodes a nuclear protein that contains a plant-specific conserved domain and physically interacts with LAX1. We propose that LAX2 is a novel factor that acts together with LAX1 in rice to regulate the process of AM formation.

A dominant mutation of TWISTED DWARF 1 encoding an alpha-tubulin protein causes severe dwarfism and right helical growth in rice.

Dwarfism is a common type of mutation in many plant species. The pathways and factors regulating biosynthesis and signaling of several plant growth regulators have been clarified through analyses of dwarf mutants in rice, Arabidopsis, pea, and maize. However, the genetic mechanisms controlling dwarfism are not well characterized, and the causal genes underlying most dwarf mutants are still uncovered. Here, we report a dominant mutant, Twisted dwarf 1-1 (Tid1-1), showing dwarfism and twisted growth in rice. Tid1-1 exhibit right helical growth of the leaves and stem and shortening of the roots. They also show an increased number of cells in the shoot apical meristem. Cells in the leaves of Tid1-1 are often ill-shapen, possibly owing to irregular cell division. Cell elongation in roots is suppressed in the elongation zone, and cells in the root apical meristem are enlarged. Map-based cloning of TID1 revealed that it encodes an alpha-tubulin protein comprising microtubules and is an ortholog of Arabidopsis LEFTY genes. Our analysis of the Tid1-1 mutant revealed that the dynamics of microtubules affects not only anisotropic growth in both dicots and monocots, but also meristematic activity and gross plant morphology.

Auxin-cytokinin interactions in the control of shoot branching.

In many plant species, the intact main shoot apex grows predominantly and axillary bud outgrowth is inhibited. This phenomenon is called apical dominance, and has been analyzed for over 70 years. Decapitation of the shoot apex releases the axillary buds from their dormancy and they begin to grow out. Auxin derived from an intact shoot apex suppresses axillary bud outgrowth, whereas cytokinin induced by decapitation of the shoot apex stimulates axillary bud outgrowth. Here we describe the molecular mechanisms of the interactions between auxin and cytokinin in the control of shoot branching.

PsRBR1 encodes a pea retinoblastoma-related protein that is phosphorylated in axillary buds during dormancy-to-growth transition.

In intact plants, cells in axillary buds are arrested at the G1 phase of the cell cycle during dormancy. In mammalian cells, the cell cycle is suppressed at the G1 phase by the activities of retinoblastoma tumor suppressor gene (RB) family proteins, depending on their phosphorylation state. Here, we report the isolation of a pea cDNA clone encoding an RB-related protein (PsRBR1, Accession No. AB012024) with a high degree of amino acid conservation in comparison with RB family proteins. PsRBR1 protein was detected as two polypeptides using an anti-PsRBR1 antibody in dormant axillary buds, whereas it was detected as three polypeptides, which were the same two polypeptides and another larger polypeptide 2 h after terminal decapitation. Both in vitro-synthesized PsPRB1 protein and lambda protein phosphatase-treated PsRBR1 protein corresponded to the smallest polypeptide detected by anti-PsRBR1 antibody, suggesting that the three polypeptides correspond to non-phosphorylated form of PsRBR1 protein, and lower- and higher-molecular mass forms of phosphorylated PsRBR1 protein. Furthermore, in vivo labeling with [32P]-inorganic phosphate indicated that PsRBR1 protein was more phosphorylated before mRNA accumulation of cell cycle regulatory genes such as PCNA. Together these findings suggest that dormancy-to-growth transition in pea axillary buds is regulated by molecular mechanisms of cell cycle control similar to those in mammals, and that the PsRBR1 protein has an important role in suppressing the cell cycle during dormancy in axillary buds.

Loss-of-function of a rice brassinosteroid biosynthetic enzyme, C-6 oxidase, prevents the organized arrangement and polar elongation of cells in the leaves and stem.

Molecular genetic and physiological studies on brassinosteroid (BR)-related mutants of dicot plants have revealed that BRs play important roles in normal plant growth and development. However, little is known about the function of BR in monocots (grasses), except for the phenotypic analysis of a rice mutant partially insensitive to BR signaling. To investigate the function of BR in monocots, we identified and characterized BR-deficient mutants of rice, BR-deficient dwarf1 (brd1). The brd1 mutants showed a range of abnormalities in organ development and growth, the most striking of which were defects in the elongation of the stem and leaves. Light microscopic observations revealed that this abnormality was primarily owing to a failure in the organization and polar elongation of the leaf and stem cells. The accumulation profile of BR compounds in the brd1 mutants suggested that these plants may be deficient in the activity of BR C-6 oxidase. Therefore, we cloned a rice gene, OsDWARF, which has a high sequence similarity to the tomato C-6 oxidase gene, DWARF. Introduction of the wild-type OsDWARF gene into brd1 rescued the abnormal phenotype of the mutants. The OsDWARF gene was expressed at a low level in all of the examined tissues, with preferential expression in the leaf sheath, and the expression was negatively regulated by brassinolide treatment. On the basis of these findings, we discuss the biological function of BRs in rice plants.

A light-switchable gene promoter system.

Regulatable transgene systems providing easily controlled, conditional induction or repression of expression are indispensable tools in biomedical and agricultural research and biotechnology. Several such systems have been developed for eukaryotes. Most of these rely on the administration of either exogenous chemicals or heat shock. Despite the general success of many of these systems, the potential for problems, such as toxic, unintended, or pleiotropic effects of the inducing chemical or treatment, can impose limitations on their use. We have developed a promoter system that can be induced, rapidly and reversibly, by short pulses of light. This system is based on the known red light-induced binding of the plant photoreceptor phytochrome to the protein PIF3 and the reversal of this binding by far-red light. We show here that yeast cells expressing two chimeric proteins, a phytochrome-GAL4-DNA-binding-domain fusion and a PIF3-GAL4-activation-domain fusion, are induced by red light to express selectable or "scorable" marker genes containing promoters with a GAL4 DNA-binding site, and that this induction is rapidly abrogated by subsequent far-red light. We further show that the extent of induction can be controlled precisely by titration of the number of photons delivered to the cells by the light pulse. Thus, this system has the potential to provide rapid, noninvasive, switchable control of the expression of a desired gene to a preselected level in any suitable cell by simple exposure to a light signal.