Somatic embryogenesis (Kamada group)
Analysis of DNA methylation and expression of embryogenesis-related genes in plants
DNA methylation is known to involve in the regulation of gene expression. In animals, the hypomethylation of DNA leads to the aberrant embryogenesis with abnormal expression of embryogenesis-related genes. On the other hand, in plants, there is limited information about the relation between DNA methylation and embryogenesis. It has been reported that DNA methylation is important in endosporem development. But it remains to be clarified whether expression of embryogenesis-related genes are controlled by DNA methylation in plants. DNA methylation is categorized into two types, maintenance methylation and de novo methylation. METHYLTRANSFERASE1(MET1) and CHROMOMETHYLASE3 (CMT3) are required for maintenance methylation. DOMAINS REARRANGED METHYLASE1 (DRM1) and DOMAINS REARRANGED METHYLASE2 (DRM2) are required for de novo methylation. Moreover, it was reported that chromatin remodeling factor DEGRESED DNA METHYLATION1 (DDM1) is able to form a complex with MET1. In this study, we investigated the relation between DNA methylation and expression of embryogenesis-related genes (e.g. LEC1, ABI3 and FUS3 in Arabidopsis, C-LEC1 and C-ABI3 in carrot). The expression of embryogenesis-related genes was examined in various tissues of the mutants defected in DNA-methylation-related genes in Arabidopsis. LEC1 showed ectopic expression in the mutants. We examined the relationship between expression of LEC1 and DNA methylation on its genomic region during embryogenesis by Southern blot analysis using DNA methylation sensitive restriction enzymes and bisulfite sequence analysis. The results suggest that DNA methylation may involve in the regulation of expression of embryogenesis-related genes and seed development in Arabidopsis. In carrot, there are some induction systems of somatic embryogenesis, which provide a large amount of synchronously developing embryos. Using the system, we tried to clarify the relationship between the expression of embryogenesis-related genes (C-LEC1 and C-ABI3) and DNA methylation on the genomic region during embryogenesis by Sourthen blot analysis, using isoschizomeric restriction enzymes and bisulfite sequencing procedure. The results suggest that DNA methylation may involve in the regulation of expression of embryogenesis-related genes in plant.
Analysis of the regulation mechanism of the phase transition from embryonic to vegetative growth in higher plants
In higher plants, transcriptional regulation by chromatin remodeling is suggested to be involved in the morphogenesis and transition of developmental phase. HDACs is one of the chromatin remodeling factors and expected to contribute to the phase transition from embryonic to vegetative growth by repressing the embryogenesis-related genes during germination. We are analyzing the involvement of the transcriptional regulation by HDACs to the phase transition from embryonic in vegetative growth.
Studiese on SET domain-containing protein genes involved in early embryogenesis in Arabidopsis thaliana
It is well known that a lot of genes are regulated temporally and spatially in embryogenesis of higher plants. However, molecular mechanisms relating to initiation of embryogenesis are still unclear. In animals, it has been reported that the gene regulation by chromatin remodeling contributes to embryogenesis. On the other hand, relationship between chromatin remodeling and initiation of embryogenesis in higher plants remains to be solved. In both mammals and plants, polycomb complexes are conserved. We are interesting in regulation of expression of embryogenesis-relatied genes by polybomb complex, and analyze double mutant, clf swn,which show somatic embryo germinated seedlings.
Search of embryo-specific histones in Arabidopsis thaliana
Histones are major components of nucleosome, the basic unit of chromatin, and contribute to form higher order structure of chromatin. Histones also involve in chromosome segregation in cell division. It is suggested that in mammals, germ cell- or embryo-specific histones exist and contribute to modify chromatin structure during early stage of embryogenesis. In higher plants, especially Lilium longiflorum and Arabidopsis thaliana, it was reported that male gamete-spesific histones exist and contribute to form highly condensed structure of chromatin during pollen formation. Based on the facts suggested in mammals, not only gamete-spesific histones, but also embryo-specific histones are expected to be expressed during embryogenesis and play an important role during embryo development. However, there is no evidence to suggest the existence of the embryo-specific histones. To identify the embryo-specific histones, we analyzed the mRNA expression of 41 genes encoding histone proteins in embryonic or vegetative tissues of Arabidopsis. HTB8 (At1g08170) which belongs to the H2B families was found to show embryo-specific expression and disappear the expression after germination. In addition, expression of HTB8 was found in stamen and pollen. These results suggest that HTB8 is expressed only in reproductive phase and embryogenesis. We are analyzing transgenic plants expressing or suppressing HTB8 to clarify the role in embryogenesis.
Flowering and biological clock (Ono group)
The FT triggers photoperiodic one-shot induction of flowering in Morning glory
Flowering is one of the most important events in the plant development, and proper day lengths accelerate flowering. Most plants need several night/day cycles to promote flowering, but in some plants flowering can be induced by only a single day treatment. One of the well-known plants showing one-shot induction of flowering is Japanese morning glory (Pharbitis nil cv. Violet), which have been studied for searching of floral stimulus. Recently, several studies using Arabidopsis assumed that FT mRNA is a main floral stimulus. Then, we used P. nil to study the role of FT as a floral stimulus in the photoperiodic one-shot induction of flowering. We isolated two FT homologs, PnFT LEAF-TYPE and PnFT APEX-TYPE, expressed in response to the inductive dark period at cotyledons and shoot apex, respectively. Over-expression of PnFT LEAF-TYPE (PnFTL) caused precocious flower formation, and RNAi plants completely abolished one-shot induction of flowering. Moreover, flowering was also observed when RNAi plants were grafted on WT plant as shoot receptor. These results indicate, in addition to meristem reactivity for induction of flowering, strict regulation of PnFTL expression is essential for one-shot induction of flowering. Moreover, the grafting experiments suggest that PnFTL mRNA is not main floral stimulus in Pharbitis nil.
On the participation of flowering and circadian clock-related genes to the photoperiodic induction of flowering in Pharbitis nil
We isolated full length cDNA clones of genes to be involved in the photoperiodic induction of flowering, between photoperiodic signal perception and floral evocation, in Pharbitis nil (cv. Violet), which has special features as a model plant of short-day floral induction, using EST information of cv. TKS. We studied the participation of genes encoding photoreceptors and components of the biological clock to the photoperiodic induction of flowering (ELF3, ELF4, CCA1, TOC1, PRRs, CDFs, LUX, ZTL, FKF1, GI, CO, FT, SOC1) by expression analysis using RT-PCR during one-shot short-day (SD) treatment. We also studied the GI-CO-FT gene cascade that is largely conserved in Arabidopsis and rice. Moreover, we compared the expression of these genes among strains and natural variations, namely, Kidachi, Tendan, Nepal and Africa that show different nature in the floral induction, and determined which genes are important for one-shot SD induction of flowering in P. nil.
Structure and function of a clock-controlled gene, AtC401, related to photoperiodic induction of flowering
The PnC401 gene, isolated from the short-day plant Pharbitis nil, shows leaf-specific and circadian regulated expression during flower-inductive darkness. PnC401 encodes a protein of 665 amino acids with no similarity to sequence in the databases. However, its Arabidopsis homologue, AtC401, encodes an 832 residue protein consisting of a kinase domain of 296 residues followed by a highly homologous to PnC401 resion (named as the C401 domain). This kinase domain belongs to the SNF1/AMPK family of protein kinases that are implicated in responses to environmental stimuli. These results suggest that the C401 domain of AtC401 is a regulatory domain for the kinase activity and that AtC401 could be involved participate in the signaling pathway of the photoperiodic flower induction.
Studies on the light signaling and circadian rhythms in the regulation of flowering time
In many plant species, a major environmental cue controlling flowering time is day length (photoperiod). Recent molecular genetic studies using Arabidopsis, a facultative LDP, have identified several genes required for the day-length response, some of which encode components of light input pathways, circadian clock components, or regulatory proteins specifically involved in the regulation of flowering. Cryptochromes (crys) are the blue-light receptors known to regulate flowering time. Crys regulate flowering by resetting the circadian clock, as well as by posttranscriptional regulation of clock-controlled genes. GIGANTEA (GI) is a key regulator of flowering time, which is closely related to the circadian clock function. Mutations in the GI gene cause photoperiod-insensitive flowering and alteration of circadian rhythms. We are working on CRYs and GI genes of Pharbitis nil. Pharbitis nil Choisy cv. Violet, an absolute SDP, is ideal for the study of early events in the photoperiodic induction of flowering. Functional analysis of these genes using transgenic plants will provide clues to a better understanding of the molecular basis for photoperiodic control of flowering in higher plants.
Study on the mechanisms of floral evocation in Pharbitis nil
Transition from vegetative to reproductive phase is often regulated by environmental signals, such as day length and temperature. After floral induction, the primary shoot meristem becomes inflorescence meristems (IM) or floral meristems (FM). According to the researches using Arabidopsis and several plants, LEAFY (LFY) and TERMINAL FLOWER1 (TFL1) play important roles to decide meristem identity. Pharbitis nil cv. Violet is a typical short-day (SD) plant and it can be induced flowering by only one SD induction. Isolation of LFY homolog and TFL1 homologues from P. nil and its characterization are carrying out to reveal the mechanisms of transition from vegetative to reproductive phase at shoot apical meristem.
Innovative designing of floricultural crops by CRES-T
Pharbitis nil Choisy cv. Violet, an SDP, is ideal for studies of the early events in the photoperiodic induction of flowering because young light-grown seedlings can be induced to flower quantitatively by exposure to a single dark period of 16 hour. We tried to isolate and characterize cDNAs that were associated with photoperiodic induction of flowering. In the late Edo era (1806~1860), many mutants were isolated, and they are still preserved partly in Kyushu University. Many genetic and physiological studies of the Pharbitis nil were conducted by Japanese biologists, and recent studies revealed that most mutations were induced by En/Spm-related transposable elements, Tpn1 family. Even if the genetic modification of Pharbitis using CRES-T, it expects that it is possible to make a similar mutant. CRES-T (Chimeric REpressor gene- Silencing Technology) is a novel gene-silencing system using the chimeric transcriptional repressors, and optional transcription factor changes into the transcriptional repressor by connecting the transcription repression domain (SRDX). The chimeric repressor dominantly suppresses the target genes. The validity of the technology, which the polar determining gene discovered with a Pharbitis nil is evaluated, and we are applying for the patent inside and outside the country. Two-year ago, the project of the second period for Promotion of Basic Research Activities for Innovative Biosciences, which entitled “Innovative designing of floricultural crops by CRES-T ” has started.
Research on the development of highly effective vaccine by plastid farming
Plastid transformation has been one of the important techniques for plant biotechnology in recent years. Compared to nucleus transformation, phenotype expressions of integrated genes by plastid transformation has many advantages, especially it has been pointed out that the amount of accumulated foreign proteins is high (De Cosa et al., 2001) . We can also recognize the convenience of plastid transformation from the fact that it has no epigenetic effects such as gene silencing or position effect and it is less likely to scatter transgenic pollens to the environment because of its maternal inheritance (Bock, 2001; Ruf et al., 2007; Svab and Maliga, 2007) . Therefore, plastid transformation in plants triggers the expectation as the means of massive synthesis of biopharmaceuticals. For, compared to biochemical synthesis, it can cut the costs of production by high expression of integrated genes due to multi copies of plastid genome DNA. This highly accumulated protein is very effective in view of oral administration of biopharmaceuticals such as vaccine. For this reason, for the past several years, gene integration of antigens such as Tetanus bacillus (TetC; Tregoning et al., 2003) , Bacillus anthracis (PA; Watson et al., 2004) and Borrelia burgdorferi (Lyme disease; Glenz et al., 2004; Chebolu and Daniell, 2007) has been practiced by using tobacco, and their expression was stable. One of the immunological experiments with animals also suggested that the antigens (vaccines) produced by plastid have stable activities and induce immunoreactions. In addition to those facts, when administrating oral vaccine, we should consider degradation by digestive enzymes. To overcome this problem, we are trying to use the antigens coated by a certain protein called VLP (Virus-Like-Particles) , which derives from virus, has tolerance to digestive enzymes and is likely to be absorbed in intestinal epithelium that is the chief part of immune system. In this study, we are trying to investigate the stable expression of VLP in chloroplast, by using tobacco which is full of observations as a model plant of chloroplast transformation. The aim of this study is getting more observations for the application to edible plants, that is, practical use of oral vaccine with crops.
Circadian clock and flowering (Mizoguchi group)
Biochemical studys of circadian rhythmicity on plant
We are investigating the circadian clock in higher plants. We specially focus on the LHY and CCA1, which function as a central clock component in Arabidopsis, and examining how these genes affecting the photoperiodic flowering by using genetic and biochemical approaches. LHY and CCA1 have DNA-binding Myb domain on their N-teminus, and supposed to function as a transcription factor. They negatively regulate the expression of TOC1, which is known as another important clock component in Arabidopsis. Since TOC1 positively regulate the expression of LHY and CCA1, these three components are supposed to comprise feedback loop as a functional clock signaling. Loss-of-function mutant of lhy cca1 has unique phenotype in photoperiodic flowering. This mutant shows early flowering under short day, and late flowering under continuous light condition. Suppressor mutagenesis provided us the novel information that ELF3, MADS-box gene FLC, and also SVP must be function with LHY and CCA1. We are trying to find relationships between these factors from a protein level.
Genetic analysis of photoperiodicity and circadian rhythmicity on plants
Circadian rhythms are oscillations in the biochemical, physiological, and behavioral functions of organisms that occur with 24 hours period with no external timing cues. In higher plants, the circadian clock controls various biological processes. Although connection between circadian clocks and photoperiodic flowering has been well characterized, it has not been well investigated that circadian clock controls other outputs such as hypocotyl elongation. To reveal novel roles of circadian clock in controlling outputs, We focus on various phenomena of circadian clock related genes mutants.
Studies on environmental risk assessment of GM plants (Kamada and Ono group)
Studies on Environmental Risk Assessment of Genetically Modified Medicinal Plants, Atropa belladonna L.
The remarkable progress of biotechnology results in production of various kinds of genetically modified (GM) plants, which are expected to be cultivated for commercial use. GM medicinal plants can be used for the effective production of secondary metabolites. In spite of potential benefits of GM plants, it is required the enough examination on environmental effects before its use in the field. One of the main subjects on environmental effects of GM plants is allelopathy. In this study, as a model case of GM medicinal plants, we investigated a method for assessing allelopathic effect of wild and transformed belladonna. We made transformed belladonna as a natural event by plant regeneration from hairy roots, which were induced by direct infection of shoot cultures with Agrobacterium rhizogenes. Then we evaluated the allelopathic effect using Sandwich method developed by the National Institute for Agro-Environmental Sciences. We found that non-transformed belladonna has an allelopathic effect, and allelopathic activity of transformants is comparable to that of non-transformants. This study will help searching the appropriate method for environmental risk assessment of GM plants.