Tomohiro Imamura

A Novel Factor FLOURY ENDOSPERM2 Is Involved in Regulation of Rice Grain Size and Starch Quality

The authors clone the rice FLOURY ENDOSPERM2 (FLO2) gene; flo2 mutants have aberrant endosperm, and FLO2 overexpressors have enlarged grains. Gene expression and protein interaction studies indicate that FLO2, a novel tetratricopeptide repeat containing protein, regulates storage starch and protein gene expression in rice endosperm development and may also play a role in heat tolerance. Rice (Oryza sativa) endosperm accumulates a massive amount of storage starch and storage proteins during seed development. However, little is known about the regulatory system involved in the production of storage substances. The rice flo2 mutation resulted in reduced grain size and starch quality. Map-based cloning identified FLOURY ENDOSPERM2 (FLO2), a member of a novel gene family conserved in plants, as the gene responsible for the rice flo2 mutation. FLO2 harbors a tetratricopeptide repeat motif, considered to mediate a protein–protein interactions. FLO2 was abundantly expressed in developing seeds coincident with production of storage starch and protein, as well as in leaves, while abundant expression of its homologs was observed only in leaves. The flo2 mutation decreased expression of genes involved in production of storage starch and storage proteins in the endosperm. Differences between cultivars in their responsiveness of FLO2 expression during high-temperature stress indicated that FLO2 may be involved in heat tolerance during seed development. Overexpression of FLO2 enlarged the size of grains significantly. These results suggest that FLO2 plays a pivotal regulatory role in rice grain size and starch quality by affecting storage substance accumulation in the endosperm.

The gentian orthologs of the FT/TFL1 gene family control floral initiation in Gentiana.

Gentians are herbaceous perennials blooming in summer through autumn. Although they are popular ornamental flowers in Japan, the regulation of their timing of flowering has not been studied. We identified and characterized gentian orthologs of the Arabidopsis FT/TFL1 gene family to elucidate the mechanisms of flowering initiation. We isolated three gentian orthologs of FT and TFL1, denoted GtFT1, GtFT2 and GtTFL1. Since up-regulation of GtFT1 and GtFT2 as well as down-regulation of GtTFL1 promoted floral initiation in gentian plantlets, these genes affected floral initiation in a similar way to Arabidopsis FT and TFL1. The expression levels of GtFT1 and GtFT2 in leaves of late-flowering gentian increased prior to floral initiation, whereas GtTFL1 was highly expressed in shoot apical meristem at the vegetative stage and decreased drastically just before flowering initiation. Comparison of gene expression patterns showed that GtFT1 expression increased earlier in early-flowering than in late-flowering gentian, whereas the timing of the increase in GtFT2 expression was similar in early- and late-flowering plants. The GtTFL1 expression in early-flowering gentian was extremely low throughout the vegetative and reproductive stages. These results indicated that either the up-regulation of GtFT1 or the down-regulation of GtTFL1 may determine flowering time. Furthermore, we found that early-flowering but not late-flowering gentians have a 320 bp insertion in the promoter region of GtTFL1. Thus, the negligible expression of GtTFL1 in early-flowering lines may be due to this insertion, resulting in a shortened vegetative stage.

The Gentio-Oligosaccharide Gentiobiose Functions in the Modulation of Bud Dormancy in the Herbaceous Perennial Gentiana

This study examines the mechanisms regulating dormancy in gentians, a group of alpine perennials that produce overwintering buds. Multivariate analysis of metabolite profiles revealed that gentiobiose, a rare disaccharide, acts as a signal for dormancy release of overwintering buds through the ascorbate-glutathione cycle, a pathway that detoxifies reactive oxygen species. Bud dormancy is an adaptive strategy that perennials use to survive unfavorable conditions. Gentians (Gentiana), popular alpine flowers and ornamentals, produce overwintering buds (OWBs) that can persist through the winter, but the mechanisms regulating dormancy are currently unclear. In this study, we conducted targeted metabolome analysis to obtain clues about the metabolic mechanisms involved in regulating OWB dormancy. Multivariate analysis of metabolite profiles revealed metabolite patterns characteristic of dormant states. The concentrations of gentiobiose [β-d-Glcp-(1→6)-d-Glc] and gentianose [β-d-Glcp-(1→6)-d-Glc-(1→2)-d-Fru] significantly varied depending on the stage of OWB dormancy, and the gentiobiose concentration increased prior to budbreak. Both activation of invertase and inactivation of β-glucosidase resulted in gentiobiose accumulation in ecodormant OWBs, suggesting that gentiobiose is seldom used as an energy source but is involved in signaling pathways. Furthermore, treatment with exogenous gentiobiose induced budbreak in OWBs cultured in vitro, with increased concentrations of sulfur-containing amino acids, GSH, and ascorbate (AsA), as well as increased expression levels of the corresponding genes. Inhibition of GSH synthesis suppressed gentiobiose-induced budbreak accompanied by decreases in GSH and AsA concentrations and redox status. These results indicate that gentiobiose, a rare disaccharide, acts as a signal for dormancy release of gentian OWBs through the AsA-GSH cycle.

Comparative metabolomics of developmental alterations caused by mineral deficiency during in vitro culture of Gentiana triflora

Gentians (Gentiana triflora, G. scabra, and hybrids of the two) are mainly cultivated as ornamental flowers in Japan. Because gentians are allogamous plants, their diversity and heterozygosity have become a major problem. Recently, explants were clonally cultured to maintain genetic purity, but culture conditions have not been studied systematically, thus the essential nutrients required for gentian culture are unknown. We therefore investigated the effects of potassium (K) and phosphorus (P) deficiency in culture media. Explants grew under K or P deficiency conditions, but P deficiency caused the formation of new structures which are similar to overwintering buds. To elucidate the mechanism behind the gentian response to mineral deficiency, we performed targeted metabolome analyses using capillary electrophoresis-mass spectrometry. Multivariate analysis using metabolite profiles showed that characteristic metabolite patterns arise in response to K or P deficiency. Under P deficiency there is a severe decrease in energy metabolites, which may in turn trigger overwintering bud formation in vitro. These findings may contribute to understanding the horticultural conditions required by gentians to trigger bud formation, and may provide a new strategy for maintaining genetic purity for long periods.

NMR based structure–activity relationship analysis of an antimicrobial peptide, thanatin, engineered by site-specific chemical modification: Activity improvement and spectrum alteration

Activity improvement of an antimicrobial peptide, thanatin, has been achieved up to 4-fold higher than natural original one by site-specific chemical modifications with tert-butyl group at two cysteine residues which form an intramoleular disulfide bridge. The chemically modified thanatin (C11tBu/C18tBu) exhibited improved antimicrobial activity toward Gram-positive bacteria, Micrococcus luteus, whereas lowered activity toward Gram-negative bacteria, Escherichia coli. This finding suggests that disulfide-bridge formation is not only indispensable for exhibition of antimicrobial activity of thanatin but also closely related to the activity specificity towards bacteria. NMR analysis indicates that thanatin acts against E.coli stereospecifically by taking advantage of its C-terminal beta-hairpin structure, while the activity against M. luteus does not relate to structures and correlates very well to side-chain hydrophobicity.

Dehydrins are highly expressed in overwintering buds and enhance drought and freezing tolerance in Gentiana triflora.

Gentians, herbaceous perennials, produce overwintering buds (OWBs) to survive the cold season. Although gentians are known to have strong stress tolerances against drought, cold and freezing, the molecular mechanisms of tolerance are unclear. We explored genes more highly expressed in OWBs than in other tissues and identified two gentian orthologs of dehydrins, denoted GtDHN1 and GtDHN2. These GtDHNs possess several ABA or dehydration responsive elements. Furthermore, GtDHN1 and GtDHN2 transcripts in OWBs accumulated during the winter but decreased prior to spring, suggesting that GtDHNs may be induced by dehydration stress during cold periods and may act as a stress protectant mediated by ABA. Likewise, cultured gentian plantlets accumulated GtDHN transcripts in response to ABA as well as cold and drought stresses. Moreover, transgenic gentian plantlets overexpressing GtDHN1 or GtDHN2 showed improved cold and drought stress tolerance. Metabolome analysis revealed that major antioxidants such as glutathione and ascorbate were accumulated in all transgenic plantlets. Overexpression of GtDHNs also affected the activities of the antioxidant enzymes, ascorbate peroxidase and glutathione peroxidase. Based on the results of this study, GtDHNs are induced by ABA and dehydration stress and have an ability to alleviate dehydration stress, probably via activating antioxidant mechanisms. Accumulation of GtDHNs may be part of the strategy for winter survival of gentian OWBs.

Characterization of spermidine synthase and spermine synthase – The polyamine-synthetic enzymes that induce early flowering in Gentiana triflora

Polyamines are essential for several living processes in plants. However, regulatory mechanisms of polyamines in herbaceous perennial are almost unknown. Here, we identified homologs of two Arabidopsis polyamine-synthetic enzymes, spermidine synthase (SPDS) and spermine synthase (SPMS) denoted as GtSPDS and GtSPMS, from the gentian plant, Gentiana triflora. Our results showed that recombinant proteins of GtSPDS and GtSPMS possessed SPDS and SPMS activities, respectively. The expression levels of GtSPDS and GtSPMS increased transiently during vegetative to reproductive growth phase and overexpression of the genes hastened flowering, suggesting that these genes are involved in flowering induction in gentian plants.

Molecular Breeding of Japanese Gentians—Applications of Genetic Transformation, Metabolome Analyses, and Genetic Markers

Japanese gentians ( Gentiana triflora , Gentiana scabra , and their hybrids) are mainly used as cut or potted flowers. Breeding of gentians using conventional techniques is very slow because of their intrinsic characteristics of heterogeneity, inbreeding depression, and long life cycle. Gentians are perennial plants and usually take 2 years to flower under natural field conditions. Various molecular biological techniques, including genetic transformation methods and DNA markers, have been developed to accelerate breeding of Japanese gentians. This chapter introduces these genetic transformation techniques and molecular genetic markers and provides recent examples of how they have been used in research on, and improvement of, Japanese gentians. Recent metabolome analyses are also described that have been used to diagnose diseases and to study the physiological conditions of gentians in vivo and in vitro. The way is also discussed in which such techniques can be applied to improve Japanese gentians in the future.

Production of recombinant thanatin in watery rice seeds that lack an accumulation of storage starch and proteins.

Molecular farming is a promising method for producing materials of commercial interest. Plants can be expected to be appropriate hosts for recombinant protein production. However, production in genetically modified plants has two major challenges that must be resolved before its practical use: insufficient accumulation of products and difficulty in establishing methods for their purification. We propose a simple procedure for the production of a desired protein using watery rice seeds lacking an accumulation of storage starch and proteins, a phenotype induced by the introduction of an antisense SPK. We produced a transgenic rice plant containing a gene for an antimicrobial peptide, thanatin, together with antisense SPK. Bioassay and proteome analysis indicated that recombinant thanatin accumulated in an active form in these watery rice seeds. These results suggest that our system worked effectively for the production of thanatin. This procedure enabled easy removal of impurities and simplified the purification process compared with production in leaves. Our system may therefore be a useful technique for the production of desired materials, including proteins.

DSH5 , a dihydrosphingosine C4 hydroxylase gene family member, shows spatially restricted expression in rice and is lethal when expressed ectopically

Dihydrosphingosine C4 hydroxylase (DSH), a diiron-binding membrane enzyme, catalyzes the hydration of dihydrosphingosine and acyl-sphinganine to produce phytosphingosine and phytoceramide, respectively. Rice has two types of DSH homologs: general DSHs, namely DSH1, DSH2 and DSH4, and others that show spatial expression profiles, namely DSH3 and DSH5. The general DSHs exist in many plant species. These DSHs showed similarity in their functions and complemented the yeast sur2D mutation. In contrast, homologs of DSH3 and DSH5 were found only in monocot plants. Phylogenetic analysis placed these DSHs in different clades that are evolutionarily divergent from those of the general DSHs. DSH3 and DSH5 showed low-level expression. DSH5 expression was specifically in vascular bundle tissues. Ectopic expression of DSH5 induced a dwarf phenotype characterized by severe growth inhibition and an increase in the thickness of the leaf body caused by enlargement of bulliform cells in the leaves. However, no significant difference was observed in the amount of sphingolipid species. DSH5 did not complement the yeast sur2D mutation, implying that DSH5 has little effect on sphingolipid metabolism. These findings suggested that DSH3 and DSH5 originated and diverged in monocot plants.