Sexual reproduction and early development of flowering plants have been major issues in plant morphology. These processes occur inside the pistil tissue, enclosed by many cell layers. Sophisticated techniques are required to study these processes. Due to difficulties associated with observation of these processes, molecular mechanisms remained unknown for many years. The molecular mechanisms, however, began to be solved recently by various achievements involving fluorescent makers for cells in these processes, detailed transcriptome analyses, and in vitro systems. Japanese young researchers also contributed to advances of this filed. In the last annual meeting of the Botanical Society of Japan, we had a joint symposium with the Japanese Society of Plant Morphology. Young researchers were invited to the symposium and contributed articles to this issue. They reviewed frontiers in sexual reproduction and early development of flowering plants.

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Self-incompatibility (SI) in crucifers is controlled by a large number of haplotypes at the S-locus. Each S-haplotype encodes a pollen-borne ligand SP11 and its stigmatic receptor, SRK, and their S-haplotype-specific interaction triggers an SI response in the stigma epidermis. The SI phenotype of pollen of an S-heterozygote is determined by the relationship between the two S-haplotypes it carries, and dominant/recessive relationships are often observed between the two S-haplotypes. These dominance hierarchies are determined at the RNA level: the SP11transcript from a recessive S-haplotype is greatly diminished in the presence of a dominant S-haplotype. In addition, weshow that the 5' promoter sequence of recessive SP11 is specifically methylated in the S-heterozygote. This promoter region is essential for SP11 transcription, and its methylation occurs specifically in the anther immediately prior to the initiation of SP11 transcription. These results suggest that tissue-specific monoallelic de novo DNA methylation is involved in determining the dominance interactions in cruciferous SI. Furthermore, possible mechanisms to explain recessive SP11 allele-specific methylation will be discussed.

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Fertilization is one of the most exciting phases in all organisms possessing sex. Understanding fertilization in plants is essential for the resolution of food problems that are dependent on agricultural products. Although plant researchers have had difficulties with analyses of gamete fusion process in angiosperm fertilization for several years, recent reports are showing remarkable findings important for understanding molecular mechanics during double fertilization. In this review, I would like to discuss the nature of angiosperm gamete fusion, based on my recent study findings.

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Plant endosperm stores nutrients for the embryo development at seed maturation and germination. It also provides an important food resource for human populations; the endosperm of rice, for example, represents a large proportion of the world's food supply. Although many aspects of the biology of endosperm have been intensively investigated, such as the cell biology, genetic controls and evolution, the early stages of endosperm development are still unclear. In many species, the endosperm develops in two phases: first, a syncytial phase and then a cellular phase. At the transition point between these phases, the syncytium is divided into individual cells by cellularization. Although cytological approaches have provided a morphological characterization of this aspect of endosperm development, the molecular mechanisms governing these events remain unclear. The endosperm can show the effects of a reproductive barrier following interspecific crosses. Various hypotheses have been proposed to explain the reproductive barriers in the hybrid endosperm of interspecific and interploidy crosses. Among these is genomic imprinting, which is defined as the parent-of-origin dependent expression of selected genes and is associated with specific epigenetic mechanisms such as DNA methylation and histone modification. In Arabidopsis, recent studies have identified several imprinted genes and also the genes required for genomic imprinting. In Arabidopsis, the Polycomb group (PcG) genes, which belong to the plant Fertilization Independent Seed (FIS) class, were identified in mutants that showed spontaneous initiation of endosperm development and seed abortion. Polycomb Repressive Complex 2 (PRC2) controls early aspects of endosperm development through gene silencing by the histone H3 trimethylated at Lysine 27 (H3K27me3). Epigenetically mediated signaling pathways, controlled by DNA methylation and H3K27me3, are essential for the control of endosperm development in Arabidopsis. Here, we summarize the genomic imprinting machinery in Arabidopsis endosperm, and focus on recent advances in our understanding of the role of PcG genes on endosperm development in rice and maize.

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During embryogenesis, the basic body plan of an organism develops from a unicellular zygote. In most flowering plants, the zygote divides asymmetrically, generating two daughter cells of different fates: the small apical daughter will form the aerial organs of the plant, whereas the large basal cell will produce the below-ground root. Despite the obvious importance, how the zygote polarizes and how this asymmetry is translated to the body axis have been obscure. In this review, we summarize the recent findings of these mechanisms, mainly focusing on the molecular genetics of Arabidopsis.

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The preferential change of giant mitochondria and their nuclei in the egg cells of Pelargonium zonale Ait. was examined during megasporogenesis, megagametogenesis, and to the first zygote division stage after the fertilization by fluorescence microscopy after Technovit embedding and 4'6-diamidino-2-phenylindole (DAPI) staining, fluorometry for DNA content, using a video-intensified microscope photon-counting system (VIMPICS), and by three-dimensional reconstruction. The reproductive cells contained many small mitochondria (0.3μm in diameter) with a small amount of DNA (0.3Mbp). As the differentiation of embryo sac began, mitochondria and their nuclei in the egg cell underwent progressive enlargement and in the mature stage, made long thick strings or stack of 5~10 concentric rings (mt-complex). There were 44 mt-complexs per cell and each one contained 340-1700 Mbp DNA. The time from pollination to cell fusion was 6~9 hours and to the first zygotic division was 20~24 hours. After the fertilization, mt-complex was gradually transformed to the single ring, to the crushed-shape and finally in the 2-celled embryo, to short fine strings or small rings and the DNA amount was about 3.4Mbp. In this review, the significance of the appearance of the peculiar giant mitochondria in the egg cell were discussed.

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The preprophase band (PPB) of microtubules marks the site of the future division plane on the plant cell cortex where the cell plate will fuse during the final stage of cytokinesis. Although the PPB microtubules on the cell cortex disappear during prometaphase, some unknown positional information is considered to remain in the PPB site after the disappearance of the PPB microtubules. Recent studies have shown that cytoskeletal proteins are known to be excluded from the PPB site. These depleted zones of cytoskeletal proteins are potential candidates for a "negative memory" system formed in the PPB site. However, the processes that produce these depleted zones of the cytoskeletal proteins are still unknown. In our recent paper, we have quantified the distribution of clathrin-coated pits and vesicles as well as of secretory structures during PPB formation of epidermal cells of onion cotyledons using a combination of high-pressure freezing and dual-axis electron-tomography techniques. Our results demonstrated that the rate of clathrin-mediated endocytosis is higher in PPB regions. We postulate that the removal of some membrane proteins by endocytosis plays a role in the creation of PPB "negative memory" structures.

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The concept of the pollen-tube attractant was proposed in the late 19th century. Since then, many plant biologists tried to identify the attractant. We identified two cysteine-rich peptides (polypeptides) specifically expressed in the synergid cell as pollen-tube attractants, by using a unique plant Torenia fournieri, which possesses a protruding embryo sac (Okuda et al. 2009). There were at least two attractant peptides, and they were named as LURE1 and LURE2. LUREs were defensin-like peptides and were secreted to the micropylar end of the synergid cell. Refolded recombinant peptides showed a strong activity to attract pollen tubes. The property of the attraction activity of LUREs were similar to those of synergid cells of Torenia, as LUREs did not attract pollen tubes germinated on the medium and pollen tubes of other plant species. Moreover, pollen tube attraction was impaired when morpholino anti-sense oligos were injected into the embryo sac by using a newly developed laser-assisted microinjector. These results indicate that LUREs are true attractant derived from the synergid cell. In this review article, I will summarize identification of LUREs and will discuss future perspectives.

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Growth within plant aerial lateral organs, such as leaves and flowers, occurs for a given period of time and stops when the organs reach their final size and shape, which are highly reproducible for genetically identical organisms grown under equivalent environments. Although leaf size is at one level simply a function of cell number and size, accumulating evidence suggests that an organ-wide integration system underlies leaf organogenesis and controls leaf size. This system, which integrates cell proliferation and expansion, has been a topic of vigorous research in recent years. In our research, we have focused on the intriguing phenomenon known as "compensation." In mutants that have a severe decrease in cell number, excessive cell enlargement is induced post-mitotically; therefore, compensation itself underscores the link between cell number and size-control systems at the level of the entire organ. In a recent large-scale histologic study of mutants of the model plant Arabidopsis thaliana, we identified a large number of leaf-size mutants with various combinations of cell-number and cell-size phenotypes. Five of these mutants, fugu1-fugu5, exhibited compensation phenotypes. Here, we highlight the recent advances in our understanding of size control and the possible mechanisms of compensated cell enlargement based on our analysis of compensation-exhibiting mutants.

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As one of the cellular slime mould mutants produced with restriction enzymes mediated integration (REMI), we isolated a strain that carries an insertion mutation in LmpB gene. LmpB is a lysosomal protein and suggested to be involved in the endosome maturation. Although some regulatory components associated with endosomes are commonly found in the contractile vacuoles, LmpB has not been suggested to be involved in the process of the osmotic regulation. However, when LmpB mutant cells were placed under osmotic stress, they developed morphologically aberrant large vacuoles in the cytoplasm and the viability of the cells was significantly reduced. The result implies that LmpB is not only involved in the endosome maturation, but also involved in the function of contractile vacuoles.

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