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Black-skinned and red-fleshed grape 'Brazil' is a bud sport of rosy-skinned 'Benitaka'. 'Brazil' has a much higher anthocyanin content in the skin than that of 'Benitaka' and is characterized by the accumulation of anthocyanins in the flesh. Our genomic analysis of the VvMYBA loci, which regulate anthocyanin biosynthesis, suggested that the difference in skin and flesh color between 'Brazil' and 'Benitaka' cannot be explained by genomic alteration at the loci. Expression levels of VvMYBA1 and anthocyanin biosynthesis-related genes in skin and flesh were significantly higher in 'Brazil' than in 'Benitaka' throughout berry development. DNA methylation levels in the 3' long terminal repeat (LTR) of a retrotransposon in the upstream region of VvMYBA1

The role of MYBA transcription factors in anthocyanin biosynthesis has been established in many plants including grapevine (Vitis vinifera L.); however, not all of the target genes of MYBAs in grapevine have been elucidated. The aim of this study was therefore to identify targets of VvMYBAs by using berry skin from a red grape (Pinot Noir) and its white mutant (Pinot Blanc), and from white grapes (Italia and Muscat of Alexandria) and their red mutants (Benitaka and Flame Muscat). First, microarray and reverse-transcription real-time PCR data revealed that, in addition to the anthocyanin specific biosynthetic genes, i.e. UDP-glucose flavonoid:3-O-glucosyltransferase (UFGT) and O-methyltransferase (OMT), a gene involved in anthocyanin transfer into vacuoles, i.e. glutathione S-transferase (GST), as well as flavonoid-3’,5’-hydroxylase (F3’,5’H) were highly upregulated in red grapes. From collective findings, it is likely that these genes are probably induced only by VvMYBAs. By contrast, genes involved in upstream flavonoid biosynthesis, such as phenylalanine ammonia-lyase (PAL), chalcone synthase 3 (CHS3), flavanone-3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), and leucoanthocyanidin dioxygenase (LDOX), were expressed in white grapes and moderately upregulated in red grapes. These genes seem to be induced by both VvMYBAs and other transcription factors. The electrophoretic mobility shift assays results revealed that VvMYBA1 and/or VvMYBA2 proteins interact with the promoter region of UFGT, CHS3, and LDOX. Collectively, our findings suggest that VvMYBAs regulate the expression of all genes involved in anthocyanin biosynthesis, and that VvMYBA protein directly interacts with not only UFGT, but also CHS3 and LDOX.

To characterize post-mortem changes in fish meat quality during chilled and frozen storage, muscle toughness and the connective tissue structure of muscle were compared between two mackerel species, spotted mackerel Scomber australasicus and Pacific mackerel S. japonicus. Measurement of the breaking strength of meat slices from both non-frozen fresh fish and frozen-thawed fish revealed that spotted mackerel meat was softer than Pacific mackerel meat. Under scanning electron microscopic observation, the connective tissue of non-frozen meat of spotted mackerel was thinner than that of Pacific mackerel. In addition, the collagen content in the spotted mackerel meat was lower than that of Pacific mackerel. These findings suggest that connective tissue structure might influence muscle toughness in mackerel.

Novel compounds based on 1a were synthesized with the focus of obtaining agonists acting upon peripheral BRS-3. To identify potent anti-obesity compounds without adverse effects on the central nervous system (CNS), a carboxylic acid moiety and a labile carboxylic ester with an antedrug functionality were introduced. Through the extensive synthetic exploration and the pharmacokinetic studies of intravenous administration in mice, the ester 2b was selected owing to its most suitable pharmacological profile. In the evaluation of food intake suppression in C57BL/6N mice, 2b showed significant in vivo efficacy and no clear adverse effects on blood pressure change in dogs administered the compound by intravenous infusion.

Persimmon (Diospyros kaki Thunb) is hexaploid, and the pollination-constant, non-astringent (PCNA)/non-PCNA trait of Japanese origin is qualitatively controlled by the AST/ast alleles at a single locus and the PCNA trait is recessive to the non-PCNA trait. To avoid inbreeding depression led by repeated crosses among PCNA genotypes, non-PCNA genotypes should be used as cross parents. The marker-assisted selection system has been developed for the selection of PCNA offspring in the progeny derived from the cross of non-PCNA ‘Taigetsu’ (non-PCNA ‘Kurokuma’ × PCNA ‘Taishu’) to PCNA ‘Kanshu’. The primer pairs E8.5/E9r and 7H9F/AST-R were used for detecting the molecular markers A1 and A3, respectively, which link AST alleles. Complete agreement was found between the sequence-characterized amplified region (SCAR) marker genotype and fruit astringency phenotype of the 48 offspring. The result confirmed that the marker-assisted selection using those markers was highly practical. In a larger offspring population (522 offspring) from the same cross, offspring segregated into 100 with both markers, 162 with only A1, 179 with A3, and 81 with neither, and this segregation ratio was significantly different from 2:3:3:2, which is the segregation ratio of random chromosome assortment in autohexaploid. The percentage of offspring expected to be PCNA was 15.5% (81 of 522), which was slightly lower than 20%.

Pollination-constant, non-astringent (PCNA) type persimmons (Diospyros kaki Thunb.) steadily lose fruit astringency and are more desirable than other types of persimmons. The PCNA/non-PCNA trait is qualitatively controlled by AST/ast alleles at a single locus. Persimmons are hexaploid, and PCNA of Japanese origin and non-PCNA genotypes are denoted as aaaaaa and A-----, respectively, if AST gene denotes A and ast gene denotes a. ‘Taiten’ is an F1, non-PCNA persimmon offspring with genotype AAaaaa derived from PCNA × non-PCNA, and molecular markers linked AST alleles in ‘Taiten’ shows polymorphism as A2 and A3, so that the genotype of ‘Taiten’ can show A2A3aaaa using the alleles of molecular marker. Offspring from a backcross of ‘Taiten’ × ‘Kanshu’ (PCNA) were evaluated. The astringency type was analyzed phenotypically, by fruit sensory and brown specks tests, and genotypically by SCAR markers with PCR primer pairs (E4/A2r for detecting the SCAR marker A2 and 7H9F/AST-R for both A2 and A3). The ratio of PCNA offspring was 43/251 (17.1%), and there was discrepancy between the phenotypic evaluation and SCAR marker estimation in only 3 offspring. The ratio was not significantly different from the theoretical ratio of nulliplex in autohexaploid. The PCNA offspring segregation ratio was 36/151 (23.8%) for ‘Fuyu’ (PCNA) × ‘Taiten’ and 6/29 (20.7%) for ‘Tenjingosho’ (PCNA) × ‘Taiten’ (nearly the same ratio reported for ‘Taiten’ × ‘Kanshu’). Marker-assisted selection using SCAR markers is thus effective and practical in persimmon breeding.

Anthocyanins affect the coloration of grape berry skin, which is an important determinant for consumer choice and grape marketability. Molecular mechanisms for anthocyanin accumulation in grape skin have been intensively studied. In Vitis vinifera, two adjacent MYB transcriptional factors, VvMybA1 and VvMybA2, regulate anthocyanin biosynthesis, and these genes can be regarded as a MYB haplotype. Interspecific hybrid grape (Vitis labruscana × V. vinifera) is particularly valuable in wet climate regions, yet few studies on anthocyanin accumulation in this hybrid have been published. Using 98 F1 individuals from a V. labruscana × V. vinifera cross, we constructed the parental and consensus linkage maps with simple sequence repeat markers, and performed quantitative trait locus (QTL) analysis of anthocyanin content. Distribution of anthocyanin content showed continuous variation, which was mainly due to the effect of genotypes. We detected QTLs for anthocyanin content in linkage groups 2 (A2-QTL1 = MYB haplotype), 8 (A8-QTL2) and 14 (A14-QTL3). These results suggest that A8-QTL2 and A14-QTL could be novel loci that affect anthocyanin content in berry skin.

The discovery and optimization of a novel series of BRS-3 agonists are described. We explored a potent BRS-3 agonist with low brain penetration to avoid an adverse effect derived from central nervous system exposure. Through the derivatization process, chiral diazepines 9f and 9g were identified as possessing low brain penetration as well as potent in vitro activity against human and mouse BRS-3s.

The pollination-constant, non-astringent (PCNA) type of persimmon is ideal for production because its fruits lose astringency at harvest regardless of seed formation. The PCNA trait in Japanese persimmons is controlled by a single locus, AST, and is recessive to the non-PCNA trait. Because cultivated persimmon is hexaploid, only the homozygous genotype with six recessive alleles is PCNA. A region tightly linked to AST has been used as a DNA marker for breeding. Three non-PCNA (A) alleles have been reported. Here, we show that the region linked to AST is highly polymorphic and includes microsatellites. By analyzing the size of PCR-amplified fragments, we distinguished 12 different A alleles from 14 non-PCNA cultivars and a Chinese PCNA ‘Luotian-tianshi.’ Then, using A fragment size, we assessed A allele inheritance in six non-PCNA × PCNA populations by analyzing segregation of each A allele in a population and segregation of progeny genotypes. By using A allele segregation analysis, we were able to estimate the copy number of each A allele in five non-PCNA parents but not in ‘Amahyakume.’ By analyzing progeny genotype segregation, we were able to estimate the ‘Amahyakume’ genotype. Our approach can be used not only for the selection of PCNA individuals in populations, but also for estimation of the copy number of A alleles in a possible non-PCNA parent. This would enable us to select non-PCNA parents with fewer A alleles, which would segregate more PCNA individuals in crosses with PCNA cultivars.

Proanthocyanidins (PAs) are secondary metabolites that contribute to plant protection and crop quality. Persimmon (Diospyros kaki) has a unique characteristic of accumulating large amounts of PAs, particularly in its fruit. Normal astringent-type and mutant nonastringent-type fruits show different PA accumulation patterns depending on the seasonal expression patterns of DkMyb4, which is a Myb transcription factor (TF) regulating many PA pathway genes in persimmon. In this study, attempts were made to identify the factors involved in DkMyb4 expression and the resultant PA accumulation in persimmon fruit. Treatment with abscisic acid (ABA) and an ABA biosynthesis inhibitor resulted in differential changes in the expression patterns of DkMyb4 and PA biosynthesis in astringent-type and nonastringent-type fruits depending on the development stage. To obtain an ABA-signaling TF, we isolated a full-length basic leucine zipper (bZIP) TF, DkbZIP5, which is highly expressed in persimmon fruit. We also showed that ectopic DkbZIP5 overexpression in persimmon calluses induced the up-regulation of DkMyb4 and the resultant PA biosynthesis. In addition, a detailed molecular characterization using the electrophoretic mobility shift assay and transient reporter assay indicated that DkbZIP5 recognized ABA-responsive elements in the promoter region of DkMyb4 and acted as a direct regulator of DkMyb4 in an ABA-dependent manner. These results suggest that ABA signals may be involved in PA biosynthesis in persimmon fruit via DkMyb4 activation by DkbZIP5.

The limited number of clinically available antifungal drugs for life-threatening fungal infections has produced an increased demand for new agents. In the course of our screening for novel antifungals, we identified aminopiperidine derivatives which exhibit antifungal activities against the major pathogenic yeasts. Thin layer chromatography (TLC) analysis of the extracted non-saponifiable lipids from Candida albicans showed that these compounds inhibited the ergosterol production in the late step of the synthesis pathway. The results of an LC/Q-Tof MS analysis showed that abnormal sterols including predicted ignosterol, which is known to be accumulated in C. albicans ERG24 deleted mutant, were accumulated in C. albicans treated with one of these derivatives (Compound 1b). Furthermore, the partial disruption of the cell membrane of C. albicans treated with compound 1b was observed by electron microscopy analysis, suggesting its inhibition of ergosterol synthesis. Additionally, a genetic approach demonstrated that ERG24 gene would be responsible for the resistance of Saccharomyces cerevisiae against Compound 1b, strongly indicating that the enzyme targeted by Compound 1b is Erg24p. From all these data, we concluded that these aminopiperidine derivatives are novel antifungal compounds inhibiting C-14 reduction in the ergosterol synthesis pathway.

Pollination-constant non-astringent (PCNA) trait is desirable in persimmon production because it confers natural astringency loss in mature persimmon fruit. Expression of the PCNA trait requires six homozygous recessive PCNA (ast) alleles at the single ASTRINGENCY (AST) locus in hexaploid persimmon. When crossing non-PCNA accessions to breed PCNA offspring, knowledge of ast and non-PCNA (AST) allele dosage in the parental accessions is important, because more PCNA offspring can segregate from a non-PCNA parent with more ast and fewer AST alleles. Previously, we have demonstrated that a region linked to the AST locus has numerous fragment size polymorphisms with varying numbers of simple sequence repeats. Here, we reveal the polymorphisms in this region in a broad collection of persimmon germplasms. Among 237 accessions, we distinguished 21 AST- and 5 ast-linked fragments with different sizes. Based on the number of fragments detected per individual, we identified 21 non-PCNA accessions with three different ast alleles; by crossing these with a PCNA parent, we obtain PCNA offspring under autohexaploid inheritance. Furthermore, AST and ast allelic combination patterns in hexaploid persimmon were shown to be applicable to cultivar identification of non-PCNA accessions. We directly sequenced ast-linked fragments from 48 accessions with one-size peak of ast-linked fragment and found two distinctive groups of fragments based on single nucleotide polymorphisms. This result suggests that a bottleneck event occurred during ast allele development. We conclude that our fragment size profile can be used to accelerate PCNA breeding that uses non-PCNA parents and to study ast allele accumulation in persimmon.

A series of cis-1,2-diaminocyclohexane derivatives possessing a 6-6 fused ring for the S1 moiety were synthesized as novel factor Xa (fXa) inhibitors. The synthesis, structure-activity relationship (SAR), and physicochemical properties are reported herein, together with the discovery of compound 45c, which has potent anti-fXa activity, good physicochemical properties and pharmacokinetic (PK) profiles, including a reduced negative food effect.

Color mutations in grape berry skin are relatively frequent events, and can be easily seen in the vineyard. Both light-red-skinned ‘Ruby Okuyama’ and more intense and uniform rosy-skinned ‘Benitaka’ (Vitis vinifera L.) are bud sports of white-skinned ‘Italia’. Previously, we reported that ‘Ruby Okuyama’ was caused by the recovery of VvmybA1 expression, which may have occurred as a result of intra-LTR (long terminal repeat) recombination within a retrotransposon, Gret1. However, the molecular basis of the color recovery in ‘Benitaka’ has not been elucidated so far. Here, we found that the VvmybA1 locus of ‘Benitaka’ is heterozygous for the VvmybA1a allele (non-functional) and a novel VvmybA1BEN allele, and that VvmybA1BEN restored VvmybA1 transcripts. We hypothesized that VvmybA1BEN allele was caused by homologous recombination between VvmybA1a and VvmybA3. In addition, the content and composition of anthocyanins in berry skins differed greatly between ‘Ruby Okuyama’ and ‘Benitaka’. The levels of expression of the genes for flavonoid 3′,5′-hydroxylase (F3′5′H), O-methyltransferase (OMT), and glutathione-S-transferase (GST) were associated with differences in the anthocyanin content and composition between the two cultivars.

A special education program (Wakakusa class) has been set up for the mentally handicapped children at the school (Ninoe Public Elementary School at Tokyo Edogawa-ward) where I currently work. Registered in that class are 14 children who have features of autism, pervasive developmental disorder, and the likes, 3 mentally retarded students, and 3 pupils with Down's disease. Autism is a developmental disorder caused by the imperfect of the central nervous system that is distinguished by a characteristic triad of symptoms, namely: 1) impaired interpersonal relationship and social interaction; 2) impairments in language and communication; and 3) restricted interest and self-stimulatory behavior. However, since some peculiar cases can also be seen like the superior music sensibility of some of them, it is said that there is also an unknown field concerning the function of the brain. (Yoshihisa Abe [An Introduction to Special Needs Education] Nihon Bunkasha, 2006) In order for the autistic children to develop their Japanese communication skill, the teachers create teaching material. On the other hand, if these children find it difficult to communicate in Japanese which is their first language, I thought that learning a second language such as English would be good for them. At the same time, I thought that the process of second language acquisition may influence and hopefully, have good effect in their Japanese. Furthermore, children must be symbolic, self-determined, independent, and have the energy and vitality to keep up with the progressing globalization. That is also the makings of the foundation of international communication power. Even the children who have special needs are not an exception. It was through "Cheerful Communication Activities Made way for the Truant and the Autistic Child to be the ALT's Favorite" and "The Ability to Have Pleasant Conversation without Fear and Hesitation Obtained" (Kanji Watanabe [International and English Communication in Elementary and Junior High Schools:Issues and Perspectives] Bunkyo Gakuin University Department of Foreign Languages and Bunkyo Gakuin College Bulletin 7th issue 2007) that I gained confidence in English activities. Through this practice, I hope that each and every autistic child would be able to build interpersonal relations, pull out the initiative and confidence to communicate, and to cultivate individuality.

Color variation in grape skin is mainly determined by the quantity and composition of anthocyanins. Anthocyanin quantities are mainly determined by the myeloblastosis (MYB) diplotypes at the color locus. We investigated whether MYB diplotypes also control the anthocyanin composition. Using a population derived from an interspecific hybrid (Vitis labruscana × Vitis vinifera) cross, we performed quantitative trait locus (QTL) analyses for the ratios of tri/di-hydroxylated and methylated/non-methylated anthocyanins. We detected the color locus on LG2 as a major QTL for the ratio of tri/di-hydroxylated anthocyanins. We also found two major QTLs, the color locus on LG2 and a region close to the anthocyanin O-methyltransferase (AOMT) locus on LG1, for the ratio of methylated/non-methylated anthocyanins. Using the same population and a population from another interspecific hybrid cross, we investigated the relationship between the genotypes of the markers closest to the major QTLs, the ratios of tri/di-hydroxylated and methylated/non-methylated anthocyanins, and the expression levels of the genes for flavonoid 3′,5′-hydroxylase (F3'5'H), flavonoid 3′-hydroxylase (F3'H), and AOMT. We suggest that MYB diplotypes at the color locus affect the ratio of tri/di-hydroxylated anthocyanins through trans-regulation of F3'5'H/F3'H expression ratio. We also suggest that MYB diplotypes and the AOMT genotypes affect the ratio of methylated/non-methylated anthocyanins through the regulation of AOMT expression. Our findings provide new knowledge about the genetic control of anthocyanin composition and contribute to a better understanding of the genetic mechanisms that control grape skin color.

We investigated the expression of the five anthocyanin biosynthetic genes, chalcone synthase ( CHS ), flavanone 3-hydroxylase ( F3H ), dihydroflavonol 4-reductase ( DFR ), anthocyanidin synthase ( ANS ), and UDP-glucose : flavonoid 3-O-glucosyltransferase ( UFGluT ) during ripening in five early ripening apple cultivars (‘Iwai’, ‘Sansa’, ‘Tsugaru’, ‘Homei-Tsugaru’, and ‘Akane’), as well as under UV-B and temperature treatments. Anthocyanin biosynthesis increased progressively during ripening, with the onset of rapid accumulation occurring at 107 days after full bloom (DAFB) in the four apple cultivars except ‘Iwai’. Generally, increase in the expression levels of all five biosynthetic genes in fruit skin coincided with increase of anthocyanin concentration. When the fruit was bagged about 1 month prior to commercial harvest to prevent anthocyanin biosynthesis, the expression of CHS , ANS and UFGluT was substantially depressed in fruit skin, whereas that of all five genes including CHS , ANS and UFGluT was enhanced by UV-B and low temperature treatments with the accumulation of anthocyanins. The accumulation level of the main anthocyanin pigment, cyanidin 3-galactoside, in the fruits under UV-B and low temperature treatment, was less than that in the field-ripened fruits, while cyanidin 3-arabinoside was relatively the same level. Our results showed that UV-B and low temperature were important factors for anthocyanin accumulation in apple fruit skin by inducing the expression of the anthocyanin biosynthetic genes, especially CHS , ANS and UFGluT genes.

A series of 4-oxo-4H-pyrido[1,2-a]pyrimidine derivatives, derivatized at the 2-position with carbon-linked substituents, were synthesized and evaluated for their ability to potentiate the activity of the fluoroquinolone levofloxacin (LVFX) and the anti-pseudomonas beta-lactam aztreonam (AZT) in Pseudomonas aeruginosa. Palladium-catalyzed cross-coupling methods were applied for the incorporation of aliphatic and aromatic substituents.

Compound 7 was identified as the active metabolite of 6 by HPLC and mass spectral analysis. Modification of lead compound 7 by transformation of its N -oxide 6–6 biaryl ring system and fused aromatics produced a series of non-basic fXa inhibitors with excellent potency in anti-fXa and anticoagulant assays. The optimized compounds 73b and 75b showed sub to one digit micromolar anticoagulant activity (PTCT2). Particularly, anti-fXa activity was detected in plasma of rats orally administered with 1 mg/kg of compound 75b .

By using white-skinned cultivars (‘Italia’ and ‘Muscat of Alexandria’) and putative red-skinned sports (‘Ruby Okuyama’ and ‘Flame Muscat’) from the white cultivars, we analyzed the expression and function of a myb-related gene, VvmybA1, involved in the regulation of anthocyanin biosynthesis in grapes. A simple sequence repeat (SSR) analysis showed that ‘Ruby Okuyama’ and ‘Flame Muscat’ are true derivatives by bud mutation from ‘Italia’ and ‘Muscat of Alexandria’, respectively. The VvmybA1 transcript was detected in berry skins of ‘Ruby Okuyama’ after coloring had begun; it was not detectable in those of ‘Italia’. Three VvmybA cDNAs (VvmybA1, VvmybA2, and VvmybA3) from ‘Ruby Okuyama’ under the control of the 35S promoter were introduced into somatic embryos of ‘Kyoho’ by particle bombardment. Upon the introduction of VvmybA1, red cells were induced in the embryos, whereas the introduction of VvmybA2 or VvmybA3 failed to do so. The VvmybA1 cDNA was also shown to have the ability to induce the anthocyanin-producing cells in the skin tissues of ‘Muscat of Alexandria’. The relationship between the skin-color mutations and the detailed structure of the VvmybA1 gene is also described.

SummaryThe effects of abscisic acid (ABA) and 2,4-dichlorophenoxyacetic acid (2,4-D) on the expression of seven anthocyanin biosynthetic pathway genes in ‘Kyoho’ grape berries were investigated. In untreated berries, the expression of the UDP-glucose-flavonoid: 3-O-glucosyltransferase (UFGT) gene was detected only at 42 d after full bloom (DAB), whereas the phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR) and leucoanthocyanidin dioxygenase (LDOX) genes were expressed throughout the growing period. ABA increased anthocyanin content in the skin and the expression of PAL, CHS, CHI, DFR and UFGT genes at 7 d after treatment. In contrast, 2,4-D inhibited the accumulation of anthocyanin and the expression of all the genes examined. The results clearly show that the anthocyanin levels resulting from the application of ABA and 2,4-D were correlated with the expression of anthocyanin biosynthetic pathway genes.

Cyanidin 3-galactoside was the primary anthocyanin in red `Tsugaru' apples [Malus sylvestris (L.) Mill. var. domestica (Borkh.) Mansf.]. The concentration of cyanidin 3-galactoside in the skin decreased from 20 to 62 days after full bloom (DAFB), then increased rapidly after 104 DAFB. Small amounts of cyanidin 3-arabinoside and cyanidin 3-glucoside were detected at 122 and 133 DAFB (harvest). The expression of five anthocyanin biosynthetic genes of chalcone synthase (MdCHS), flavanone 3-hydroxylase (MdF3H), dihydroflavonol 4-reductase (pDFR), anthocyanidin synthase (MdANS), and UDP glucose-flavonoid 3-O-glucosyltransferase (pUFGluT) was examined in the skin of red and nonred apples. In general, the expression of anthocyanin biosynthetic genes in red apples was strong in juvenile and ripening stages. The expression of MdCHS, MdF3H, pDFR, and MdANS was observed before ripening stage when anthocyanin was not detected. In contrast, the expression of pUFGluT was detected in the development stage only when anthocyanin was detected. However, the expression of all five genes was observed at 20 DAFB in fruit bagged after fertilization, and anthocyanin was not detected. The expression of MdCHS, MdF3H, pDFR, and MdANS, excluding pUFGluT, was detected at 98 DAFB in fruit bagged after 30 DAFB, and anthocyanin was not detected. These results suggest that pUFGluT may be closely related to the anthocyanin expression in apple skin at the ripening stage.

Five genes of anthocyanin biosynthetic enzymes, chalcone synthase (CHS; EC 2.3.1.74), flavanone 3-hydroxylase (F3H; EC 1.14.11.9), dihydroflavonol 4-reductase (DFR; EC 1.1.1.219), anthocyanidin synthase (ANS; EC 1.14.11.X), and UDP glucose:flavonoid 3-O-glucosyltransferase (UFGluT; EC 2.4.1.X), were isolated, and their expression was investigated to elucidate the molecular mechanism for red coloration in apple 〚Malus sylvestris (L.) Mill. var. domestica (Borkh.) Mansf.〛 skin. In ‘Orin,’ a yellow apple cultivar, no significant levels of anthocyanin were detectable, whereas in ‘Jonathan’ and ‘Fuji,’ both red apple cultivars, anthocyanin concentrations increased during fruit development. At the ripe stage, the level of anthocyanin concentration was about three times higher in ‘Jonathan’ than in ‘Fuji.’ The accumulation of transcripts for the five genes was induced at the later developmental stages in all three cultivars. The levels for the expression of the five genes basically corresponded to the anthocyanin concentrations; that is, the induction of the genes in ‘Orin’ was less pronounced, and that in ‘Fuji’ and ‘Jonathan’ was notable, with much higher expression levels in ‘Jonathan’ than in ‘Fuji’. These results indicate that the five genes are coordinately expressed during fruit development and that their levels of expression are positively related to the degree of anthocyanin concentration. This is the first report that characterizes the relationship between the expression of anthocyanin biosynthetic genes and apple fruit coloration.

Partial cDNAs of myb-related regulatory genes were isolated from the tetraploid Kyoho grape ( Vitis labruscana: V. labrusca x V. vinifera) and the expression patterns of the corresponding genes were studied. Since MybA gene expression is closely related to coloring and/or ripening of the berry (expression increases strongly with the commencement of coloring and berry softening, and is detected only in berry skin and flesh), full-length cDNAs for the gene were isolated from a mature-berry cDNA library. Three different species of MybA were identified from the cDNA sequences. Delivery of these cDNAs to somatic embryos of grape led to the induction of reddish-purple spots and UDP-glucose:flavonoid 3- O-glucosyltransferase (UFGT) gene expression in non-colored embryos. The UFGT transcript was not detected in control embryos, while other structural genes for anthocyanin biosynthesis were expressed in both control and pigmented embryos. In addition, introduction of the UFGT gene induced the same reddish-purple spots in embryos. In contrast, treatment with the leucoanthocyanidin dioxygenase (LDOX) gene failed to induce these spots. Our results strongly suggest that MybA genes are involved in the regulation of anthocyanin biosynthesis in the grape via expression of the UFGT gene.

Six partial cDNAs for cell wall degradation-related enzymes, xyloglucan endo-transglycosylase (XET), polygalacturonase (PG), pectin methylesterase (PME), pectate lyase (PL), cellulase (Cel), and β- d -galactosidase (GAL), were isolated from a veraison-specific subtractive library made from Kyoho grape (Vitis labruscana) berries. During the development of Kyoho grape berries, the expression of these genes was analyzed. Among the genes analyzed, XET gene expression was closely related to berry softening; slight XET gene expression was detected before veraison and was markedly increased at veraison (the stage of the onset of berry softening). In addition, the expression of the gene was berry specific. In the other genes, except for PL, however, no expression was detected during the berry development; PL was detected only after the coloring stage began. These observations suggest that XET plays an important role in grape berry softening. To obtain further information about this gene, a full-length cDNA clone (VXET 1) encoding XET was isolated from a cDNA library of Kyoho grape berries and characterized. The VXET 1 was 1326 bp in length and contained a 5′-untranslated region of 67 bp, an open reading frame of 873 bp, and a 3′-untranslated region of 386 bp. The deduced amino acid sequence of the VXET 1 showed 73.5% identity with the corresponding XET (NXG 1) from nasturtium (Tropaeolum majus) that has been shown to have endo-glucanase activity. These findings suggest that the VXET 1 product cleaves a cellulose–xyloglucan network of cell wall and induces the softening of Kyoho grape berries at veraison.

SummaryThe expression of five anthocyanin biosynthetic genes of chalcone synthase (MdCHS). flavanane 3-hydroxylase (MdF3H). dihydroflavonol 4-reducase (pDFR), anthocyanidin synthase (MdANS) and UDP glucose-flavonoid 3-O-glucosyltransferase (pUFGluT) was investigated during fruit development in the skin of non-red ‘Mutsu’ apples (Malus sylvestris (L) Mill. van domestica (Borkh) Mansf.). MdCHS, MdF3H and pDFR appeared in the skin of non-bagged fruit from 81 d after full bloom (DAFB) to 167 DAFB, but MdANS and pUFGluT did not appear. When the fruit was bagged during fruit development and unbagged before the maturation, anthocyanin was produced in the exposed portion but not in the shaded portion. All five genes including MdANS and pUFGluT were detected in the exposed portion at 165 DAFB. In the shaded portion, MdANS was detected, but pUFGluT was not. The skin colour of the shaded portion after unbagging in the bagged fruit was more yellow than that in non-bagged fruit. This result suggests that the yellowing...

The expression of the UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT) gene has been shown to be critical for anthocyanin biosynthesis in the grape berry. Using white cultivars and bud sports with red skin, we examined the expression of seven anthocyanin biosynthetic genes including the UFGT gene and compared the coding/promoter sequences of the UFGT gene. Northern blot analysis showed that the seven anthocyanin biosynthetic genes were expressed coordinately at higher levels in the red-skin sports than in the white-skin progenitors of the sports. It was especially notable that UFGT gene expression was detected only in the red-skin sports and Kyoho. However, there were no differences in either coding or promoter sequences between Italia (Vitis vinifera) and its red-skin sport Ruby Okuyama, or between Muscat of Alexandria (V. vinifera) and the red-skin sport Flame Muscat. From these findings, the phenotypic change from white to red in the sports is thought to be the result of a mutation in a regulatory gene controlling the expression of UFGT.

Electrofusion was conducted to use the embryogenic callus cells of satsuma mandarin (Citrus unshiu cv. ‘Juman’ unshiu) and mesophyll cells of yuzu (C. junos) and lemon (C.limon cv. ‘Eureka’ lemon). Two plants were regenerated from the combination of satsuma mandarin and yuzu, and one plant from that of satsuma mandarin and lemon. The plants had 18 chromosomes (2n=2×=18 in each parent), and showed the same nuclear rDNA fragment pattern as that of the mesophyll parent. The mitochondrial DNA analysis showed the presence of specific yuzu or lemon bands in addition to specific satsuma mandarin bands. From these results with morphological evidence, the regenerated plants were considered as cybrids with recombined mitochondrial DNA.

Transgenic plants of trifoliate orange (Poncirus trifoliata Raf.) possessing chimeric genes, a promoter of cauliflower mosaic virus (CaMV) 35S RNA and structural gene of rolC of the Ri plasmid (35S-rolC) or promoter region (5' upstream of non-coding DNA) of rolC and the structural gene of rolC (rolC-rolC) were obtained by Agrobacterium-mediated transformation. The stable integration of the genes was confirmed by polymerase chain reaction (PCR) and Southern blot analyses. The transformed plants are dwarfed, but the degree of dwarfing differed among individual plants and between plants with 35S-rolC and those with rolC-rolC. The internodal lengths of the transformants with 35S-rolC were much shorter than those of nontransformed control plants. Eighteen out of 20 transformants with 35S-rolC were 40% shorter than the control. The internodal length of plants with rolC-rolC, in contrast, ranged from 10-120% of control plants. Almost all transformants possessed higher rooting abilities than did the control plants.

A white-wine grape, Pinot Blanc, is thought to be a white-skinned mutant of a red-wine grape, Pinot Noir. Pinot Noir was heterozygous for VvmybA1. One allele was the non-functional VvmybA1a, and the other was the functional VvmybA1c. In Pinot Blanc, however, only VvmybA1a was observed, and the amount of VvmybA1 DNA in Pinot Blanc was half that in Pinot Noir. These findings suggest that deletion of VvmybA1c from Pinot Noir resulted in Pinot Blanc.

RCC2, cDNA clone encoding rice class-I chitinase, was introduced into trifoliate orange (Poncirus trifoliata Raf.). Chitinase activity of the transformed plants was higher than that of the non-transformed plants. The introduced gene was not detected in the scions grafted onto the transformed plants. The effect of the introduction of foreign gene into rootstocks on the scions was discussed.

To produce citrus triploids through somatic hybridization, protoplasts isolated from nucellar calli of 'Juman' satsuma mandarin, 'Ohmishima' navel orange or 'Trovita' sweet orange were fused electrically with protoplasts from mesophyll cells of haploid strains of Clementine (No. 1 and No. 2) ; three combinations (satsuma mandarin + haploid No. 1, navel orange+haploid No. 2, and sweet orange+haploid No. 1) were practically tried.Chromosome observations of root tip cells showed that the regenerated plants were diploid (2 n=18) or triploid (2 n=27). In each combination, triploid plants were observed. Hybridity of the plants was checked by RAPD-PCR method. Triploid plants produced in this study contained both parent-specific bands, indicating that the triploid plants are somatic hybrids.

Crosses between satsuma mandarins and tetraploid ponkan were made to produce triploid hybrid cultivars.Triploid embryos were obtained from the crosses using three seed parents, 'Ootsu No. 4', 'Aoshima' and 'Imamura', which produced comparatively small numbers of embryo per seed. Of the three, the frequency of triploid embryo production per fruit was highest in 'Aoshima'. The frequency of triploid embryo production per seed in undeveloped seeds was higher than that in fully developed seeds. By the embryo rescue technique, triploid plants were obtained efficiently from the embryos of undeveloped seeds. Tetraploid plants were also obtained from fully developed seed of 'Ootsu No. 4' and 'Aoshima'.RAPD analysis showed that all the triploid plants were hybrids and the tetraploid plants were also hybrids or natural auto-tetraploids. Two hybrid embryos in one seed with identical banding RAPD patterns on agarose gels, indicate that the embryos are derived from the same single egg cell.

Chloroplast DNA (cpDNA) diversity was studied in Citrus, Poncirus and Fortunella, based on restriction fragment length polymorphism (RFLP). Total genomic DNA was digested with restriction endonucleases singly or in combination, Southern blotted, and hybridized with a Nicotiana tabacum cpDNA probe. According to the fragment patterns, Citrus was divided into four groups; mandarin/citron, pummelo, Yuzu, and lime. The fragment patterns of mandarin/citron group (mandarin, citron and tangor) differed from those of pummelo group (pummelo, sweet orange, sour orange, lemon, ' grapefruit, Kunenbo, Hassaku, Natsudaidai, Sudachi, Hyuganatsu and Iyo) in Pst I, Pst I/Pvu II, Pst I/Kpn I and Pst I/ Sac I digestion. The fragment patterns of lime were identical to pummelo group in Pst I and Pst I/ Sac I digestion, but differed in Pst I/Pvu II and Pst I/Kpn I digestion. Yuzu was not distinguishable from mandarin/citron group in the fragment patterns of Pst I, Pst I/ Pvu II and Pst I/ Kpn I digestion, but was distinguishable in that of Pst I/ Sac I digestion. The fragment patterns of Poncirus and Fortunella were always identical to those of mandarin/citron group.

Kiwifruit (Actinidia chinensis) and trifoliate orange (Poncirus trifoliata) were trans- formed with a chemically synthesized gene encoding the human epidermal growth factor (hEGF) under the control of the cauliflower mosaic virus 35S RNA gene promoter. The introduced gene (s) were expressed in the young leaves of the regenerated plants. The highest hEGF peptide content so far detected, by a sandwich enzyme-linked immunosor- bent assay for hEGF, was about 65 and 113 pg•mg-1 soluble proteins in kiwifruit and trifoliate orange leaves, respectively.This is the first demonstration of the production of human bioactive peptides, non- fused with other proteins, in fruit trees and is suggesting the possibility of producing some useful components in fruits by using transformation technique.

Electrofusion was conducted to combine Citrus unshiu cv. ‘Juman’ unshiu protoplasts isolated from embryogenic callus with C. sinensis cv. ‘F. N. Washington’ navel orange mesophyll protoplasts. One plant was regenerated from the fusion products. The plant has 18 chromosomes (2n=18 in each parent), and it showed the same nuclear rDNA fragment pattern as that of C. sinensis. Whereas, chloroplast and mitochondrial DNA analyses showed that the plant contained C. unshiu chloroplast and mitochondrial genomes. From these results, the regenerated plant was confirmed as a cybrid having C. sinensis nuclear genome and C. unshiu cytoplasmic genome.