The MKK3 MAPK cascade integrates temperature and after-ripening signals to modulate seed germination.

The timing of seed germination is controlled by the combination of internal dormancy and external factors. Temperature is a major environmental factor for seed germination. The permissive temperature range for germination is narrow in dormant seeds and expands during after-ripening (AR) (dormancy release). Quantitative trait loci analyses of preharvest sprouting in cereals have revealed that MKK3, a mitogen-activated protein kinase (MAPK) cascade protein, is a negative regulator of grain dormancy. Here, we show that the MAPKKK19/20-MKK3-MPK1/2/7/14 cascade modulates the germination temperature range in Arabidopsis seeds by elevating the germinability of the seeds at sub- and supraoptimal temperatures. The expression of MAPKKK19 and MAPKKK20 is induced around optimal temperature for germination in after-ripened seeds but repressed in dormant seeds. MPK7 activation depends on the expression levels of MAPKKK19/20, with expression occurring under conditions permissive for germination. Abscisic acid (ABA) and gibberellin (GA) are two major phytohormones which are involved in germination control. Activation of the MKK3 cascade represses ABA biosynthesis enzyme gene expression and induces expression of ABA catabolic enzyme and GA biosynthesis enzyme genes, resulting in expansion of the germinable temperature range. Our data demonstrate that the MKK3 cascade integrates temperature and AR signals to phytohormone metabolism and seed germination.

Wheat Cybrid Plants, OryzaWheat, Regenerated from Wheat-Rice Hybrid Zygotes via in Vitro Fertilization System Possess Wheat-Rice Hybrid Mitochondria.

Hybridization generates biodiversity, and wide hybridization plays a pivotal role in enhancing and broadening the useful attributes of crops. The hybridization barrier between wheat and rice, the two most important cereals, was recently overcome by in vitro production of allopolyploid wheat-rice hybrid zygotes, which can develop and grow into mature plants. In the study, genomic sequences and compositions of the possible hybrid plants were investigated through short- and long-read sequencing analyses and fluorescence in situ hybridization (FISH)-based visualization. The possible hybrid possessed whole wheat nuclear and cytoplasmic DNAs and rice mitochondrial (mt) DNA, along with variable retention rates of rice mtDNA ranging from 11% to 47%. The rice mtDNA retained in the wheat cybrid, termed Oryzawheat, can be transmitted across generations. In addition to mitochondrial hybridization, translocation of rice chromosome 1 into wheat chromosome 6A was detected in a F1 hybrid individual. OryzaWheat can provide a new horizon for utilizing inter-subfamily genetic resources among wheat and rice belonging to different subfamilies, Pooideae and Ehrhartoideae, respectively.

An ABCB transporter regulates anisotropic cell expansion via cuticle deposition in the moss Physcomitrium patens.

Anisotropic cell expansion is crucial for the morphogenesis of land plants, as cell migration is restricted by the rigid cell wall. The anisotropy of cell expansion is regulated by mechanisms acting on the deposition or modification of cell wall polysaccharides. Besides the polysaccharide components in the cell wall, a layer of hydrophobic cuticle covers the outer cell wall and is subjected to tensile stress that mechanically restricts cell expansion. However, the molecular machinery that deposits cuticle materials in the appropriate spatiotemporal manner to accommodate cell and tissue expansion remains elusive. Here, we report that PpABCB14, an ATP-binding cassette transporter in the moss Physcomitrium patens, regulates the anisotropy of cell expansion. PpABCB14 localized to expanding regions of leaf cells. Deletion of PpABCB14 resulted in impaired anisotropic cell expansion. Unexpectedly, the cuticle proper was reduced in the mutants, and the cuticular lipid components decreased. Moreover, induced PpABCB14 expression resulted in deformed leaf cells with increased cuticle lipid accumulation on the cell surface. Taken together, PpABCB14 regulates the anisotropy of cell expansion via cuticle deposition, revealing a regulatory mechanism for cell expansion in addition to the mechanisms acting on cell wall polysaccharides.

P-MIRU, a Polarized Multispectral Imaging System, Reveals Reflection Information on the Biological Surface.

Reflection light forms the core of our visual perception of the world. We can obtain vast information by examining reflection light from biological surfaces, including pigment composition and distribution, tissue structure and surface microstructure. However, because of the limitations in our visual system, the complete information in reflection light, which we term 'reflectome', cannot be fully exploited. For example, we may miss reflection light information outside our visible wavelengths. In addition, unlike insects, we have virtually no sensitivity to light polarization. We can detect non-chromatic information lurking in reflection light only with appropriate devices. Although previous studies have designed and developed systems for specialized uses supporting our visual systems, we still do not have a versatile, rapid, convenient and affordable system for analyzing broad aspects of reflection from biological surfaces. To overcome this situation, we developed P-MIRU, a novel multispectral and polarization imaging system for reflecting light from biological surfaces. The hardware and software of P-MIRU are open source and customizable and thus can be applied for virtually any research on biological surfaces. Furthermore, P-MIRU is a user-friendly system for biologists with no specialized programming or engineering knowledge. P-MIRU successfully visualized multispectral reflection in visible/non-visible wavelengths and simultaneously detected various surface phenotypes of spectral polarization. The P-MIRU system extends our visual ability and unveils information on biological surfaces.

Phylogenetic distribution and expression pattern analyses identified a divergent basal body assembly protein involved in land plant spermatogenesis.

Land plant spermatozoids commonly possess characteristic structures such as the spline, which consists of a microtubule array, the multilayered structure (MLS) in which the uppermost layer is a continuum of the spline, and multiple flagella. However, the molecular mechanisms underpinning spermatogenesis remain to be elucidated. We successfully identified candidate genes involved in spermatogenesis, deeply divergent BLD10s, by computational analyses combining multiple methods and omics data. We then examined the functions of BLD10s in the liverwort Marchantia polymorpha and the moss Physcomitrium patens. MpBLD10 and PpBLD10 are required for normal basal body (BB) and flagella formation. Mpbld10 mutants exhibited defects in remodeling of the cytoplasm and nucleus during spermatozoid formation, and thus MpBLD10 should be involved in chromatin reorganization and elimination of the cytoplasm during spermiogenesis. We identified orthologs of MpBLD10 and PpBLD10 in diverse Streptophyta and found that MpBLD10 and PpBLD10 are orthologous to BLD10/CEP135 family proteins, which function in BB assembly. However, BLD10s evolved especially quickly in land plants and MpBLD10 might have acquired additional functions in spermatozoid formation through rapid molecular evolution.

Expression of Genes from Paternal Alleles in Rice Zygotes and Involvement of OsASGR-BBML1 in Initiation of Zygotic Development.

Upon fertilization in angiosperms, one sperm cell fuses with the egg cell to produce a zygote, and, via karyogamy, the parental genetic information is combined to form the diploid zygotic genome. Recently, analyses with parentally imbalanced rice zygotes indicated that parental genomes are utilized synergistically in zygotes with different functions, and that genes transcribed from the paternal or maternal allele might play important roles in zygotic development. Herein, we first conducted single nucleotide polymorphism-based mRNA-sequencing using intersubspecific rice zygotes. Twenty-three genes, with paternal allele-specific expression in zygotes, were identified, and, surprisingly, their allele dependencies in the globular-like embryo tended to be biallelic. This suggests that the paternal-dependent expression of these genes is temporary, occurring during the early stages of zygote development. Of the 23 genes, we focused on Oryza sativa Apospory-specific Genome Region (ASGR)-BABY-BOOM LIKE (BBML) 1 (OsASGR-BBML1), presumed to encode an AP2-transcription factor, due to its reported role in zygotic development. Interestingly, ectopic expression of OsASGR-BBML1 in egg cells induced nuclear and cell divisions, indicating that exogenously expressed OsASGR-BBML1 converts the proliferation status of the egg cell from quiescent to active. In addition, the suppression of the function of OsASGR-BBML1 and its homologs in zygotes resulted in the developmental arrest, suggesting that OsASGR-BBML1 possesses an important role in initiating zygotic development. Monoallelic or preferential gene expression from the paternal genome in the zygote might be a safety mechanism allowing egg cells to suppress the gene expression cascade toward early embryogenesis that is normally triggered by fusion with a sperm cell.

Protocol: an improved method for inducing sporophyte generation in the model moss Physcomitrium patens under nitrogen starvation.

BACKGROUND: Land plants exhibit a haplodiplontic life cycle, whereby multicellular bodies develop in both the haploid and diploid generations. The early-diverging land plants, known as bryophytes, have a haploid-dominant life cycle, in which a short-lived multicellular body in the diploid generation, known as the sporophyte, develops on the maternal haploid gametophyte tissues. The moss Physcomitrium (Physcomitrella) patens has become one of the most powerful model systems in evolutionary plant developmental studies. To induce diploid sporophytes of P. patens, several protocols are implemented. One of the conventional approaches is to grow approximately one-month-old gametophores for another month on Jiffy-7 pellets made from the peat moss that is difficult to fully sterilize. A more efficient method to obtain all tissues throughout the life cycle should accelerate studies of P. patens. RESULTS: Here, we investigated the effect of nitrogen conditions on the growth and development of P. patens. We provide an improved protocol for the sporophyte induction of P. patens using a BCD-based solid culture medium without Jiffy-7 pellets, based on the finding that the formation of gametangia and subsequent sporophytes is promoted by nitrogen-free growth conditions. The protocol consists of two steps; first, culture the protonemata and gametophores on nitrogen-rich medium under continuous light at 25 °C, and then transfer the gametophores onto nitrogen-free medium under short-day and at 15 °C for sporophyte induction. The protocol enables to shorten the induction period and reduce the culture space. CONCLUSIONS: Our more efficient and shortened protocol for inducing the formation of sporophytes will contribute to future studies into the fertilization or the diploid sporophyte generation of P. patens.

Genome and transcriptome analyses reveal genes involved in the formation of fine ridges on petal epidermal cells in Hibiscus trionum.

Hibiscus trionum, commonly known as the 'Flower of an Hour', is an easily cultivated plant in the Malvaceae family that is widespread in tropical and temperate regions, including drylands. The purple base part of its petal exhibits structural colour due to the fine ridges on the epidermal cell surface, and the molecular mechanism of ridge formation has been actively investigated. We performed genome sequencing of H. trionum using a long-read sequencing technology with transcriptome and pathway analyses to identify candidate genes for fine structure formation. The ortholog of AtSHINE1, which is involved in the biosynthesis of cuticular wax in Arabidopsis thaliana, was significantly overexpressed in the iridescent tissue. In addition, orthologs of AtCUS2 and AtCYP77A, which contribute to cutin synthesis, were also overexpressed. Our results provide important insights into the formation of fine ridges on epidermal cells in plants using H. trionum as a model.

Lanthanum Supplementation Alleviates Tomato Root Growth Suppression under Low Light Stress.

Supplementation with rare earth elements (REEs) such as lanthanum and cerium has been shown to promote plant elongation and/or increase crop yields. On the other hand, there are reports that REE supplementation of plants has no such effect. The appropriate modes for REE utilization and the underlying mechanism are not fully understood. In this study, we investigated how REE supplementation of plants under low light stress affects plant growth and gene expression. Under low light stress conditions, tomato root elongation was observed to be reduced by about half. This suppression of root elongation was found to be considerably alleviated by 20 mM lanthanum ion supplementation. This effect was plant-species-dependent and nutrient-condition-dependent. Under low light stress, the expression of the genes for phytochrome-interacting factor, which induces auxin synthesis, and several auxin-synthesis-related proteins were markedly upregulated by lanthanum ion supplementation. Thus, we speculate that REE supplementation of plants results in auxin-induced cell elongation and alleviates growth suppression under stress conditions.

Effect of Paternal Genome Excess on the Developmental and Gene Expression Profiles of Polyspermic Zygotes in Rice.

Polyploid zygotes with a paternal gamete/genome excess exhibit arrested development, whereas polyploid zygotes with a maternal excess develop normally. These observations indicate that paternal and maternal genomes synergistically influence zygote development via distinct functions. In this study, to clarify how paternal genome excess affects zygotic development, the developmental and gene expression profiles of polyspermic rice zygotes were analyzed. The results indicated that polyspermic zygotes were mostly arrested at the one-cell stage after karyogamy had completed. Through comparison of transcriptomes between polyspermic zygotes and diploid zygotes, 36 and 43 genes with up-regulated and down-regulated expression levels, respectively, were identified in the polyspermic zygotes relative to the corresponding expression in the diploid zygotes. Notably, OsASGR-BBML1, which encodes an AP2 transcription factor possibly involved in initiating rice zygote development, was expressed at a much lower level in the polyspermic zygotes than in the diploid zygotes.

TRANSNAP: a web database providing comprehensive information on Japanese pear transcriptome.

Japanese pear (Pyrus pyrifolia) is a major fruit tree in the family Rosaceae and is bred for fruit production. To promote the development of breeding strategies and molecular research for Japanese pear, we sequenced the transcripts of Japanese pear variety 'Hosui'. To exhaustively collect information of total gene expression, RNA samples from various organs and stages of Japanese pear were sequenced by three technologies, single-molecule real-time (SMRT) sequencing, 454 pyrosequencing, and Sanger sequencing. Using all those reads, we determined comprehensive reference sequences of Japanese pear. Then, their protein sequences were predicted, and biological functional annotations were assigned. Finally, we developed a web database, TRANSNAP (http://plantomics.mind.meiji.ac.jp/nashi), which is the first web resource of Japanese pear omics information. This database provides highly reliable information via a user-friendly web interface: the reference sequences, gene functional annotations, and gene expression profiles from microarray experiments. In addition, based on sequence comparisons among Japanese, Chinese and European pears, similar protein sequences among the pears and species-specific proteins in Japanese pear can be quickly and efficiently identified. TRANSNAP will aid molecular research and breeding in Japanese pear, and its information is available for comparative analysis among other pear species and families.

Physcomitrella MADS-box genes regulate water supply and sperm movement for fertilization.

MIKC classic (MIKCC)-type MADS-box genes encode transcription factors that function in various developmental processes, including angiosperm floral organ identity. Phylogenetic analyses of the MIKCC-type MADS-box family, including genes from non-flowering plants, suggest that the increased numbers of these genes in flowering plants is related to their functional divergence; however, their precise functions in non-flowering plants and their evolution throughout land plant diversification are unknown. Here, we show that MIKCC-type MADS-box genes in the moss Physcomitrella patens function in two ways to enable fertilization. Analyses of protein localization, deletion mutants and overexpression lines of all six genes indicate that three MIKCC-type MADS-box genes redundantly regulate cell division and growth in the stems for appropriate external water conduction, as well as the formation of sperm with motile flagella. The former function appears to be maintained in the flowering plant lineage, while the latter was lost in accordance with the loss of sperm.