Identification of a novel viral factor inducing tumorous symptoms by disturbing vascular development in planta.

Plant viruses induce various disease symptoms that substantially impact agriculture, but the underlying mechanisms of viral disease in plants are poorly understood. Kobu-sho is a disease in gentian that shows gall formation with ectopic development of lignified cells and vascular tissues such as xylem. Here, we show that a gene fragment of gentian Kobu-sho-associated virus, which is designated as Kobu-sho-inducing factor (KOBU), induces gall formation accompanied by ectopic development of lignified cells and xylem-like tissue in Nicotiana benthamiana. Transgenic gentian expressing KOBU exhibited tumorous symptoms, confirming the gall-forming activity of KOBU. Surprisingly, KOBU expression can also induce differentiation of an additional leaf-like tissue on the abaxial side of veins in normal N. benthamiana and gentian leaves. Transcriptome analysis with Arabidopsis thaliana expressing KOBU revealed that KOBU activates signaling pathways that regulate xylem development. KOBU protein forms granules and plate-like structures and co-localizes with mRNA splicing factors within the nucleus. Our findings suggest that KOBU is a novel pleiotropic virulence factor that stimulates vascular and leaf development. IMPORTANCE While various mechanisms determine disease symptoms in plants depending on virus-host combinations, the details of how plant viruses induce symptoms remain largely unknown in most plant species. Kobu-sho is a disease in gentian that shows gall formation with ectopic development of lignified cells and vascular tissues such as xylem. Our findings demonstrate that a gene fragment of gentian Kobu-sho-associated virus (GKaV), which is designated as Kobu-sho-inducing factor, induces the gall formation accompanied by the ectopic development of lignified cells and xylem-like tissue in Nicotiana benthamiana. The molecular mechanism by which gentian Kobu-sho-associated virus induces the Kobu-sho symptoms will provide new insight into not only plant-virus interactions but also the regulatory mechanisms underlying vascular and leaf development.

Sweat-gland carcinoma with neuroendocrine differentiation (SCAND): a clinicopathologic study of 13 cases with genetic analysis.

Low-grade neuroendocrine carcinoma of the skin (LGNECS) was proposed in 2017 as a new primary cutaneous neoplasm with neuroendocrine differentiation; however, it is not yet well known due to its rarity. Herein, we perform a detailed clinicopathologic analysis of 13 cases as well as panel DNA sequencing in three cases. The study included 12 males and 1 female with a median age of 71 (43-85) years. All lesions occurred on the ventral trunk. The mean tumor size was 2.2 (0.8-11.0) cm. The histopathology resembled that of well-differentiated neuroendocrine tumors (NETs) in other organs, but intraepidermal pagetoid spreading was seen in 8 (61.5%) cases and stromal mucin deposits in 4 (30.8%). Immunoreactivity for CK7, CK19, EMA, BerEP4, CEA, chromogranin A, synaptophysin, INSM1, GCDFP15, GATA3, ER, and bcl-2 were present in varying degrees in all tested cases. PTEN c.165-1G>A splice site mutation was detected by panel sequencing in one case, and GATA3 P409fs*99 and SETD2 R1708fs*4 in another case. Lymph node metastasis was seen significantly in cases with tumor size >2.0 cm [8/8 (100%) vs. 1/5 (20%)]. All three cases with size >3.0 cm were in unresectable advanced-stage [3/3 (100%) vs. 1/10 (10%)], and two of the three patients succumbed to the disease. The two cases of death revealed mild nuclear atypia (mitosis: 1/10 HPFs) and moderate nuclear atypia (2/10 HPFs). Thus, tumor size would be a better prognostic factor than nuclear atypia, mitotic count, and Ki67 index, unlike in NETs. These clinicopathologic and immunohistochemical features would represent the characteristics as skin adnexal tumors with apocrine/eccrine differentiation rather than NETs; therefore, we rename it as sweat-gland carcinoma with neuroendocrine differentiation (SCAND).

Post-transcriptional gene silencing of the chalcone synthase gene CHS causes corolla lobe-specific whiting of Japanese gentian.

MAIN CONCLUSION: Post-transcriptional gene silencing of the chalcone synthase gene CHS specifically suppresses anthocyanin biosynthesis in corolla lobes and is responsible for the formation of a stripe type bicolor in Japanese gentian. The flower of Japanese gentian is a bell-shaped corolla composed of lobes and plicae, which is painted uniformly blue. However, the gentian cultivar 'Hakuju' shows bicolor phenotype (blue-white stripe corolla), in which anthocyanin accumulation is suppressed only in corolla lobes. Expression analysis indicated that steady-state levels of chalcone synthase (CHS) transcripts were remarkably reduced in corolla lobes compared with plicae during petal pigmentation initiation. However, no significant difference in expression levels of other flavonoid biosynthetic structural and regulatory genes was detected in its lobes and plicae. On feeding naringenin in white lobes, anthocyanin accumulation was recovered. Northern blotting probed with CHS confirmed the abundant accumulation of small RNAs in corolla lobes. Likewise, small RNA-seq analysis indicated that short reads from its lobes were predominantly mapped onto the 2nd exon region of the CHS gene, whereas those from the plicae were scarcely mapped. Subsequent infection with the gentian ovary ringspot virus (GORV), which had an RNA-silencing activity, showed the recovery of partial pigmentation in lobes. Hence, these results strongly suggested that suppressing anthocyanin accumulation in the lobes of bicolored 'Hakuju' was attributed to the specific degradation of CHS mRNA in corolla lobes, which was through post-transcriptional gene silencing (PTGS). Herein, we revealed the molecular mechanism of strip bicolor formation in Japanese gentian, and showed that PTGS of CHS was also responsible for flower color pattern in a floricultural plant other than petunia and dahlia.

Anthocyanin regulatory and structural genes associated with violet flower color of Matthiola incana.

MAIN CONCLUSION: MiMYB1 and MibHLH2 play key roles in anthocyanin biosynthesis in Matthiola incana flowers. We established a transient expression system using Turnip mosaic virus vector in M. incana. Garden stock (Matthiola incana (L.) R. Br.) is a popular flowering plant observed from winter to spring in Japan. Here we observed that anthocyanin accumulation in 'Vintage Lavender' increased with flower development, whereas flavonol accumulation remained constant throughout flower development. We obtained five transcription factor genes, MiMYB1, MibHLH1, MibHLH2, MiWDR1, and MiWDR2, from M. incana floral cDNA contigs. Yeast two-hybrid analyses revealed that MiMYB1 interacted with MibHLH1, MibHLH2, and MiWDR1, but MiWDR2 did not interact with any transcription factor. Expression levels of MiMYB1 and MibHLH2 increased in petals during floral bud development. Their expression profiles correlated well with the temporal profiles of MiF3'H, MiDFR, MiANS, and Mi3GT transcripts and anthocyanin accumulation profile. On the other hand, MibHLH1 was expressed weakly in all organs of 'Vintage Lavender'. However, high expression levels of MibHLH1 were detected in petals of other cultivars with higher levels of anthocyanin accumulation than 'Vintage Lavender'. MiWDR1 and MiWDR2 maintained constant expression levels in petals during flower development and vegetative organs. Transient MiMYB1 expression in 1-month-old M. incana seedlings using a Turnip mosaic virus vector activated transcription of the endogenous anthocyanin biosynthetic genes MiF3'H, MiDFR, and MiANS and induced ectopic anthocyanin accumulation in leaves. Therefore, MiMYB1 possibly interacts with MibHLH2 and MiWDR1, and this trimeric protein complex activates the transcription of anthocyanin biosynthetic genes in M. incana flowers. Moreover, MibHLH1 acts as an enhancer of anthocyanin biosynthesis with the MiMYB1-MibHLH2-MiWDR1 complex. This study revealed the molecular mechanism involved in the regulation of anthocyanin accumulation levels in M. incana flowers.

Inhibition of post-transcriptional gene silencing of chalcone synthase genes in petunia picotee petals by fluacrypyrim.

In petals of picotee petunia (Petunia hybrida) cultivars, margin-specific post-transcriptional gene silencing (PTGS) of chalcone synthase A (CHSA) inhibits anthocyanin biosynthesis, resulting in marginal white tissue formation. In this study, we found that a low molecular mass compound, fluacrypyrim, inhibits PTGS of CHSA, and we explored the site-specific PTGS mechanism of operation. Fluacrypyrim treatment abolished the picotee pattern and eliminated site-specific differences in the levels of anthocyanin-related compounds, CHSA expression, and CHSA small interfering RNA (siRNA). In addition, fluacrypyrim abolished the petunia star-type pattern, which is also caused by PTGS of CHSA. Fluacrypyrim treatment was effective only at the early floral developmental stage and predominantly eliminated siRNA derived from CHS genes; i.e. siRNA derived from other genes remained at a comparable level. Fluacrypyrim probably targets the induction of PTGS that specifically operates for CHS genes in petunia picotee flowers, rather than common PTGS maintenance mechanisms that degrade mRNAs and generate siRNA. Upon treatment, the proportion of colored tissue increased due to a shift of the border between white and colored sites toward the margin in a time- and dose-dependent manner. These findings imply that the fluacrypyrim-targeted PTGS induction is completed gradually and its strength is attenuated from the margins to the center of petunia picotee petals.

Clinical Findings of a Cantilever Iliac Bone Graft for Secondary Correction of Cleft Lip-Nose Deformities.

The authors performed a cantilever iliac bone graft for the secondary correction of severe cleft lip-nose deformities after the completion of growth. For the purpose of clarifying effects of the cantilever iliac bone grafts and the adverse events with regard to their time course changes after this procedure, the authors retrospectively surveyed long-term morphologic changes in 65 cleft lip, alveolus, and palate patients in whom cleft lip-nose deformities were treated with a cantilever iliac bone graft (age at surgery: 14-45 years old). All postsurgical documents of facial photographs and radiologic images were reviewed to evaluate the effects and adverse events. The main adverse events were deviations of the apex of the nose, excess resorption of the grafted iliac bone, protruding deformations of the grafted iliac bone at the root of the nose, and fracture of the grafted iliac bone. Additional surgery was necessary in 10.7% of patients. Postsurgical changes in facial profiles became favorable, measured on lateral view of cephalometric radiography, achieving morphologic improvements. A cantilever iliac bone graft was effective for improving nasal deformities in cleft lip, alveolus, and palate patients, although the counter measures should be taken to these adverse events.

Low-Grade Neuroendocrine Carcinoma of the Skin (Primary Cutaneous Carcinoid Tumor) as a Distinctive Entity of Cutaneous Neuroendocrine Tumors: A Clinicopathologic Study of 3 Cases With Literature Review.

There is scarcity of information on primary cutaneous low-grade neoplasms commonly known as carcinoid tumors, owing to their rarity. The authors present 3 cases that were named "low-grade neuroendocrine carcinoma of the skin" (LGNECS). These occurred in the dermis and subcutis of the anterior chest or the inguinal region in the elderly. Histologically, the tumors showed infiltrating proliferation of nests of various sizes, with low-grade neuroendocrine cytologic features but without mucin production. All cases exhibited varying degrees of intraductal tumor components. On immunohistochemical examination, these tumors expressed estrogen receptor alpha, progesterone receptor, androgen receptor, gross cystic disease fluid protein 15, mammaglobin, and GATA3 as well as neuroendocrine markers. Although a literature review revealed 8 additional possible cases with no evidence of other diseases, it was difficult to determine if these were true cases of LGNECS, because of the limited information available. Based on its characteristic histologic features and immunoprofile, it can be proposed designating LGNECS as a distinct entity among cutaneous neuroendocrine tumors. Otherwise, such tumors could be misdiagnosed as mammary carcinomas (particularly when involving the skin of the breast) or as metastatic visceral neuroendocrine tumors of the skin.

RNA-seq-based evaluation of bicolor tepal pigmentation in Asiatic hybrid lilies (Lilium spp.).

BACKGROUND: Color patterns in angiosperm flowers are produced by spatially and temporally restricted deposition of pigments. Identifying the mechanisms responsible for restricted pigment deposition is a topic of broad interest. Some dicots species develop bicolor petals, which are often caused by the post-transcriptional gene silencing (PTGS) of chalcone synthase (CHS) genes. An Asiatic hybrid lily (Lilium spp.) cultivar Lollypop develops bicolor tepals with pigmented tips and white bases. Here, we analyzed the global transcription of pigmented and non-pigmented tepal parts from Lollypop, to determine the main transcriptomic differences. RESULTS: De novo assembly of RNA-seq data yielded 49,239 contigs (39,426 unigenes), which included a variety of novel transcripts, such as those involved in flavonoid-glycosylation and sequestration and in regulation of anthocyanin biosynthesis. Additionally, 1258 of the unigenes exhibited significantly differential expression between the tepal parts (false discovery rates <0.05). The pigmented tepal parts accumulated more anthocyanins, and unigenes annotated as anthocyanin biosynthesis genes (e.g., CHS, dihydroflavonol 4-reductase, and anthocyanidin synthase) were expressed 7-30-fold higher than those in non-pigmented parts. These results indicate that the transcriptional regulation of biosynthesis genes is more likely involved in the development of bicolor lily tepals rather than the PTGS of CHS genes. In addition, the expression level of a unigene homologous to LhMYB12, which often regulates full-tepal anthocyanin pigmentation in lilies, was >2-fold higher in the pigmented parts. Thus, LhMYB12 should be involved in the transcriptional regulation of the biosynthesis genes in bicolor tepals. Other factors that potentially suppress or enhance the expression of anthocyanin biosynthesis genes, including a WD40 gene, were identified, and their involvement in bicolor development is discussed. CONCLUSIONS: Our results indicate that the bicolor trait of Lollypop tepals is caused by the transcriptional regulation of anthocyanin biosynthesis genes and that the transcription profile of LhMYB12 provides a clue for elucidating the mechanisms of the trait. The tepal transcriptome constructed in this study will accelerate investigations of the genetic controls of anthocyanin color patterns, including the bicolor patterns, of Lilium spp.

Functional characterization of duplicated B-class MADS-box genes in Japanese gentian.

KEY MESSAGE: The heterodimer formation between B-class MADS-box proteins of GsAP3a and GsPI2 proteins plays a core role for petal formation in Japanese gentian plants. We previously isolated six B-class MADS-box genes (GsAP3a, GsAP3b, GsTM6, GsPI1, GsPI2, and GsPI3) from Japanese gentian (Gentiana scabra). To study the roles of these MADS-box genes in determining floral organ identities, we investigated protein-protein interactions among them and produced transgenic Arabidopsis and gentian plants overexpressing GsPI2 alone or in combination with GsAP3a or GsTM6. Yeast two-hybrid and bimolecular fluorescence complementation analyses revealed that among the GsPI proteins, GsPI2 interacted with both GsAP3a and GsTM6, and that these heterodimers were localized to the nuclei. The heterologous expression of GsPI2 partially converted sepals into petaloid organs in transgenic Arabidopsis, and this petaloid conversion phenomenon was accelerated by combined expression with GsAP3a but not with GsTM6. In contrast, there were no differences in morphology between vector-control plants and transgenic Arabidopsis plants expressing GsAP3a or GsTM6 alone. Transgenic gentian ectopically expressing GsPI2 produced an elongated tubular structure that consisted of an elongated petaloid organ in the first whorl and stunted inner floral organs. These results imply that the heterodimer formation between GsPI2 and GsAP3a plays a core role in determining petal and stamen identities in Japanese gentian, but other B-function genes might be important for the complete development of petal organs.

Isolation and characterization of the C-class MADS-box gene involved in the formation of double flowers in Japanese gentian.

BACKGROUND: Generally, double-flowered varieties are more attractive than single-flowered varieties in ornamental plants. Japanese gentian is one of the most popular floricultural plants in Japan, and it is desirable to breed elite double-flowered cultivars. In this study, we attempted to characterize a doubled-flower mutant of Japanese gentian. To identify the gene that causes the double-flowered phenotype in Japanese gentian, we isolated and characterized MADS-box genes. RESULTS: Fourteen MADS-box genes were isolated, and two of them were C-class MADS-box genes (GsAG1 and GsAG2). Both GsAG1 and GsAG2 were categorized into the PLE/SHP subgroup, rather than the AG/FAR subgroup. In expression analyses, GsAG1 transcripts were detected in the second to fourth floral whorls, while GsAG2 transcripts were detected in only the inner two whorls. Transgenic Arabidopsis expressing GsAG1 lacked petals and formed carpeloid organs instead of sepals. Compared with a single-flowered gentian cultivar, a double-flowered gentian mutant showed decreased expression of GsAG1 but unchanged expression of GsAG2. An analysis of the genomic structure of GsAG1 revealed that the gene had nine exons and eight introns, and that a 5,150-bp additional sequence was inserted into the sixth intron of GsAG1 in the double-flowered mutant. This insert had typical features of a Ty3/gypsy-type LTR-retrotransposon, and was designated as Tgs1. Virus-induced gene silencing of GsAG1 by the Apple latent spherical virus vector resulted in the conversion of the stamen to petaloid organs in early flowering transgenic gentian plants expressing an Arabidopsis FT gene. CONCLUSIONS: These results revealed that GsAG1 plays a key role as a C-functional gene in stamen organ identity. The identification of the gene responsible for the double-flowered phenotype will be useful in further research on the floral morphogenesis of Japanese gentian.

Molecular characterization of mutations in white-flowered torenia plants.

BACKGROUND: Torenia (Torenia fournieri Lind.) is a model plant increasingly exploited in studies in various disciplines, including plant engineering, biochemistry, physiology, and ecology. Additionally, cultivars with different flower colors have been bred and made commercially available. Flower color in torenia is mainly attributed to the accumulation of anthocyanins, but the molecular mechanisms inducing flower color mutations in torenia have not been well elucidated. In this study, we therefore attempted to identify the cause of white coloration in torenia by comparing the white-flowered cultivar Crown White (CrW) with Crown Violet (CrV), a violet-flowered variety. RESULTS: In an expression analysis, no flavanone 3-hydroxylase (TfF3H) transcript accumulation was detected in CrW petals. Sequence analyses revealed that a novel long terminal repeat (LTR)-type retrotransposable element, designated as TORE1 (Torenia retrotransposon 1), is inserted into the 5'-upstream region of the TfF3H gene in CrW. A transient expression assay using torenia F3H promoters with or without TORE1 insertion showed that the TORE1 insertion substantially suppressed F3H promoter activity, suggesting that this insertion is responsible for the absence of F3H transcripts in white petals. Furthermore, a transformation experiment demonstrated that the introduction of a foreign gentian F3H cDNA, GtF3H, into CrW was able to recover pink-flower pigmentation, indicating that F3H deficiency is indeed the cause of the colorless flower phenotype in CrW. Detailed sequence analysis also identified deletion mutations in flavonoid 3'-hydroxylase (TfF3'H) and flavonoid 3',5'- hydroxylase (TfF3'5'H) genes, but these were not directly responsible for white coloration in this cultivar. CONCLUSIONS: Taken together, a novel retrotransposable element, TORE1, inserted into the F3H 5'-upstream region is the cause of deficient F3H transcripts in white-flowered torenia, thereby leading to reduced petal anthocyanin levels. This is the first report of a retrotransposable element involved in flower color mutation in the genus Torenia.

Nanog promotes osteogenic differentiation of the mouse mesenchymal cell line C3H10T1/2 by modulating bone morphogenetic protein (BMP) signaling.

How the pluripotency of stem cells is maintained and the role of transcription factors in this maintenance remain major questions. In the present study, in order to clarify the mechanism underlying the pluripotency of stem cells for the advancement of regenerative medicine, we examined the effect of forced Nanog expression in mesenchymal cells, with a particular focus on osteogenic differentiation. The human mesenchymal stromal cells (hMSCs) or mouse mesenchymal cell line C3H10T1/2 cells were transduced with the Nanog gene or control green fluorescent protein (GFP) gene by using retrovirus vectors. Short-term, forced Nanog gene expression had few effects on the terminal osteogenic differentiation of either hMSCs or C3H10T1/2 cells. To determine its long-term effects, we established C3H10T1/2 cells expressing Nanog constitutively. Constitutive Nanog expression strongly induced osteogenic differentiation of C3H10T1/2 cells. In regard to cell proliferation, constitutive Nanog expression only repressed the proliferation of the cells treated with rhBMP-2. Moreover, Nanog also had the potential to promote the proliferation of C3H10T1/2 cells in the absence of rhBMP-2. Constitutive Nanog expression enhanced phosphorylation of Smad1/5/8 and suppressed Cdk4 and cyclinD1. The promoter activities of both the osteocalcin and Id-1 genes were activated in cells expressing Nanog constitutively. To identify downstream molecules of Nanog involved in the promotion of osteogenic differentiation, we performed a DNA microarray analysis and discovered that NFATc1 was one of the downstream effectors of Nanog. These results indicate that Nanog functions as a modulator of BMP signaling in C3H10T1/2 cells probably through a genome reprogramming process.

Heterologous expression of gentian MYB1R transcription factors suppresses anthocyanin pigmentation in tobacco flowers.

KEY MESSAGE: Single-repeat MYB transcription factors, GtMYB1R1 and GtMYB1R9 , were isolated from gentian. Overexpression of these genes reduced anthocyanin accumulation in tobacco flowers, demonstrating their applicability to modification of flower color. RNA interference (RNAi) has recently been used to successfully modify flower color intensity in several plant species. In most floricultural plants, this technique requires prior isolation of target flavonoid biosynthetic genes from the same or closely related species. To overcome this limitation, we developed a simple and efficient method for reducing floral anthocyanin accumulation based on genetic engineering using novel transcription factor genes isolated from Japanese gentians. We identified two single-repeat MYB genes--GtMYB1R and GtMYB1R9--predominantly expressed in gentian petals. Transgenic tobacco plants expressing these genes were produced, and their flowers were analyzed for flavonoid components and expression of flavonoid biosynthetic genes. Transgenic tobacco plants expressing GtMYB1R1 or GtMYB1R9 exhibited significant reductions in floral anthocyanin accumulation, resulting in white-flowered phenotypes. Expression levels of chalcone isomerase (CHI), dihydroflavonol 4-reductase (DFR), and anthocyanidin synthase (ANS) genes were preferentially suppressed in these transgenic tobacco flowers. A yeast two-hybrid assay demonstrated that both GtMYB1R1 and GtMYB1R9 proteins interacted with the GtbHLH1 protein, previously identified as an anthocyanin biosynthesis regulator in gentian flowers. In addition, a transient expression assay indicated that activation of the gentian GtDFR promoter by the GtMYB3-GtbHLH1 complex was partly canceled by addition of GtMYB1R1 or GtMYB1R9. These results suggest that GtMYB1R1 and GtMYB1R9 act as antagonistic transcription factors of anthocyanin biosynthesis in gentian flowers. These genes should consequently be useful for manipulating anthocyanin accumulation via genetic engineering in flowers of other floricultural plant species.

Recent advances in reconstructive surgery: head and neck reconstruction.

Although advances in radiotherapy and chemotherapy for cancers of the head and neck have been remarkable, surgical resection followed by reconstructive surgery is still the mainstay of treatment. Of the reconstructive procedures, microsurgical tissue transfer has been considered the standard method for restoring postoperative functions and morphology. In this review article, we discuss the history of reconstructive surgery for treating cancers of the head and neck, current problems, and future challenges.

Clinical factors associated with postoperative complications and the functional outcome in mandibular reconstruction.

BACKGROUND: Few studies have performed a multiple factor analysis to assess the factors associated with successful mandibular reconstructions in a large number of subjects. The purpose of this study is to evaluate the functional outcome in mandibular reconstruction by means logistic regression analysis. METHODS: Since April 2005 to September 2009, 126 patients underwent segmental resection of the mandible for cancer ablation and mandibular reconstruction with free flaps at 6 Japanese institutions. The patients' charts were reviewed retrospectively. Twelve patients were excluded for the reconstruction was with double flaps, or they went under secondary reconstruction. With logistic regression analysis in 114 subjects, we assessed multiple factors influencing postoperative speech intelligibility, feeding ability, and postoperative complications of mandibular reconstruction. RESULTS: The use of a reconstruction plate with a soft-tissue free flap only was showed to have a deleterious effect on postoperative feeding. The strong association in the level of statistical significance between the use of a reconstruction plate with soft-tissue free flaps only and the occurrences of major complications was indicated. It was also statistically revealed that the postoperative presence of opposing teeth contributed to both speech intelligibility and oral intake. CONCLUSIONS: In our research, osteocutaneous flaps were superior to reconstruction plates with soft-tissue free flaps regard to the postoperative feeding ability and major complication rate.

Construction of the first genetic linkage map of Japanese gentian (Gentianaceae).

BACKGROUND: Japanese gentians (Gentiana triflora and Gentiana scabra) are amongst the most popular floricultural plants in Japan. However, genomic resources for Japanese gentians have not yet been developed, mainly because of the heterozygous genome structure conserved by outcrossing, the long juvenile period, and limited knowledge about the inheritance of important traits. In this study, we developed a genetic linkage map to improve breeding programs of Japanese gentians. RESULTS: Enriched simple sequence repeat (SSR) libraries from a G. triflora double haploid line yielded almost 20,000 clones using 454 pyrosequencing technology, 6.7% of which could be used to design SSR markers. To increase the number of molecular markers, we identified three putative long terminal repeat (LTR) sequences using the recently developed inter-primer binding site (iPBS) method. We also developed retrotransposon microsatellite amplified polymorphism (REMAP) markers combining retrotransposon and inter-simple sequence repeat (ISSR) markers. In addition to SSR and REMAP markers, modified amplified fragment length polymorphism (AFLP) and random amplification polymorphic DNA (RAPD) markers were developed. Using 93 BC1 progeny from G. scabra backcrossed with a G. triflora double haploid line, 19 linkage groups were constructed with a total of 263 markers (97 SSR, 97 AFLP, 39 RAPD, and 30 REMAP markers). One phenotypic trait (stem color) and 10 functional markers related to genes controlling flower color, flowering time and cold tolerance were assigned to the linkage map, confirming its utility. CONCLUSIONS: This is the first reported genetic linkage map for Japanese gentians and for any species belonging to the family Gentianaceae. As demonstrated by mapping of functional markers and the stem color trait, our results will help to explain the genetic basis of agronomic important traits, and will be useful for marker-assisted selection in gentian breeding programs. Our map will also be an important resource for further genetic analyses such as mapping of quantitative trait loci and map-based cloning of genes in this species.

Isolation and characterization of GtMYBP3 and GtMYBP4, orthologues of R2R3-MYB transcription factors that regulate early flavonoid biosynthesis, in gentian flowers.

Flavonoids are one of the major plant pigments for flower colour. Not only coloured anthocyanins, but also co-pigment flavones or flavonols, accumulate in flowers. To study the regulation of early flavonoid biosynthesis, two R2R3-MYB transcription factors, GtMYBP3 and GtMYBP4, were identified from the petals of Japanese gentian (Gentiana triflora). Phylogenetic analysis showed that these two proteins belong to the subgroup 7 clade (flavonol-specific MYB), which includes Arabidopsis AtMYB12, grapevine VvMYBF1, and tomato SlMYB12. Gt MYBP3 and Gt MYBP4 transcripts were detected specifically in young petals and correlated with the profiles of flavone accumulation. Transient expression assays showed that GtMYBP3 and GtMYBP4 enhanced the promoter activities of early biosynthetic genes, including flavone synthase II (FNSII) and flavonoid 3'-hydroxylase (F3'H), but not the late biosynthetic gene, flavonoid 3',5'-hydroxylase (F3'5'H). GtMYBP3 also enhanced the promoter activity of the chalcone synthase (CHS) gene. In transgenic Arabidopsis, overexpression of Gt MYBP3 and Gt MYBP4 activated the expression of endogenous flavonol biosynthesis genes and led to increased flavonol accumulation in seedlings. In transgenic tobacco petals, overexpression of Gt MYBP3 and Gt MYBP4 caused decreased anthocyanin levels, resulting in pale flower colours. Gt MYBP4-expressing transgenic tobacco flowers also showed increased flavonols. As far as is known, this is the first functional characterization of R2R3-MYB transcription factors regulating early flavonoid biosynthesis in petals.

Synthesis and biological evaluation of truncated α-galactosylceramide derivatives focusing on cytokine induction profile.

A series of truncated analogs of α-galactosylceramide with altered ceramide moiety was prepared, and evaluated for Th2-biased response in the context of IL-4/IFN-γ ratio. Phytosphingosine-modified analogs including cyclic, aromatic and ethereal compounds as well as the C-glycoside analog of OCH (2) with their cytokine inducing profile are disclosed.

Establishment of an efficient transformation method of garden stock (Matthiola incana) using a callus formation chemical inducer.

Matthiola incana is an important floricultural plant that blooms from winter to spring, and had been desired to be established a transformation system. This study successfully obtained stable transgenic plants from M. incana. We used Agrobacterium tumefaciens harboring a binary vector containing the ß-glucuronidase gene (GUS) under the control of cauliflower mosaic virus 35S promoter to evaluate the transformation frequency of M. incana. We observed that cocultivation with the A. tumefaciens strain GV3101 for 5 days effectively enhanced the infection frequency, assessed through a transient GUS expression area in the seedling. Furthermore, the addition of 100 µM acetosyringone was necessary for Agrobacterium infection. However, we could not obtain transgenic plants on a shoot formation medium supplemented with 1 mg l-1 6-benzyladenine (BA). For callus formation from the leaf sections, a medium supplemented with 1-50 µM fipexide (FPX), a novel callus induction chemical, was employed. Then, the callus formation was observed after 2 weeks, and an earlier response was detected than that in the BA medium (4-6 weeks). Results also showed that cultivation in a selection medium supplemented with 12.5 µM FPX obtained hygromycin-resistant calli. Thus, this protocol achieved a 0.7% transformation frequency. Similarly, progenies from one transgenic line were observed on the basis of GUS stains on their leaves, revealing that the transgenes were also inherited stably. Hence, FPX is considered a breakthrough for establishing the transformation protocol of M. incana, and its use is proposed in recalcitrant plants.

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.