Institute of Fruit Tree and Tea Science

The color of grape skins is determined by the accumulation of red plant pigments called anthocyanins. White cultivars of grape are thought to have arisen from dif-ferent red cultivars by independent muta-tions (1), but the molecular bases of these color mutations are unknown. Myb-related genes (such as VlmybA1-1, VlmybA1-2, and VlmybA2) regulate anthocyanin biosynthe-sis in Kyoho, a black-skinned cultivar of Vitis labruscana (2). We show that a retrotransposon-induced mutation in VvmybA1, a homolog of VlmybA1-1, is associated with the loss of pigmentation in white cultivars of V. vinifera. Two red-skinned cultivars of V. vinif-era, Ruby Okuyama (Ru) and Flame Mus-cat (Fl), are derived by bud mutation from the white-skinned cultivars Italia (It) and Muscat of Alexandria (Al), respectively. Using VlmybA1-1 from Kyoho as a probe, we detected two transcripts in white-skinned cultivars and three in the red-skinned sports. Sequencing identified transcripts VvmybA2 (DNA Data Bank of Japan accession no. AB097924) and VvmybA3 (AB097925) in all four cultivars and VvmybA1 (AB097923) in the red cul-tivars (Fig. 1A). In the other cultivars examined, VvmybA1 transcript was also detected only in the colored ones (Fig.1B). VvmybA1 cDNA induced red pigmentation when introduced into the skin tissues of white grapes (3). Genomic clones for VvmybA1 indicated that VvmybA1 is homozygous (VvmybA1a, AB111100) in It, but heterozygous in Ru

An HPLC analysis was performed on the concentrations of flavonoids in 42 species and cultivars of the Citrus genus and those of two Fortunella and one Poncirus species according to the classification system established by Tanaka. The composition of 8 flavanones and 9 flavone/ols for these species was determined in the albedo, flavedo, segment epidermis and juice vesicle tissues, and those in the fruit and peel tissues were calculated from the composition data of the tissues. A principal component analysis showed that such neohesperidosyl flavonoids as neoeriocitrin, naringin, neohesperidin, and rhoifolin had large factor loading values in the first principal component for each tissue. The flavonoid composition of citrus fruits was approximately the same within each section of Tanaka's system, except for the species in the Aurantium section and those with a peculiar flavonoid composition such as Bergamot (C. bergamia), Marsh grapefruit (C. paradisi), Sour orange (C. aurantium), and Shunkokan (C. shunkokan). The Aurantium section included both naringin-rich and hesperidin-rich species.

Red coloration of apple (Malus domestica) skin is an important determinant of consumer preference and marketability. Anthocyanins are responsible for this coloration, and their accumulation is positively correlated with the expression level of anthocyanin biosynthetic genes. Regulation of expression of these genes is believed to be controlled by MYB transcription factors, and the MYB transcription factors involved in the activation of anthocyanin biosynthetic genes have been isolated in various plants. In the present study, we isolated and characterized a MYB transcription factor gene (MdMYBA) from apple skin. Characterization of MdMYBA demonstrated that (i) MdMYBA expression was specifically regulated depending on the tissue and cultivar/ species; (ii) its expression level was much higher in a deep-red cultivar ('Jonathan') than in a pale-red cultivar ('Tsugaru'); (iii) when cauliflower mosaic virus 35S::MdMYBA was introduced into the cotyledons of apple seedlings by means of a transient assay, reddish-purple spots were induced, and MdMYBA also induced anthocyanin accumulation in reproductive tissues of transgenic tobacco; (iv) the expression of MdMYBA was induced by UV-B irradiation and lowtemperature treatment, both of which are known to be important in the promotion of anthocyanin accumulation in apple skin; (v) MdMYBA bound specifically to an anthocyanidin synthase (MdANS) promoter region in a gel-shift assay; and (vi) MdMYBA was mapped to the near region of the BC226-STS (a 1 ) marker for the red skin color locus (R f ). These results suggest that MdMYBA is a key regulatory gene in anthocyanin biosynthesis in apple skin.

The relationship between carotenoid accumulation and the expression of carotenoid biosynthetic genes during fruit maturation was investigated in three citrus varieties, Satsuma mandarin (Citrus unshiu Marc.), Valencia orange (Citrus sinensis Osbeck), and Lisbon lemon (Citrus limon Burm.f.). We cloned the cDNAs for phytoene synthase (CitPSY), phytoene desaturase (CitPDS), zeta-carotene (car) desaturase (CitZDS), carotenoid isomerase (CitCRTISO), lycopene beta-cyclase (CitLCYb), beta-ring hydroxylase (CitHYb), zeaxanthin (zea) epoxidase (CitZEP), and lycopene epsilon-cyclase (CitLCYe) from Satsuma mandarin, which shared high identities in nucleotide sequences with Valencia orange, Lisbon lemon, and other plant species. With the transition of peel color from green to orange, the change from beta,epsilon-carotenoid (alpha-car and lutein) accumulation to beta,beta-carotenoid (beta-car, beta-cryptoxanthin, zea, and violaxanthin) accumulation was observed in the flavedos of Satsuma mandarin and Valencia orange, accompanying the disappearance of CitLCYe transcripts and the increase in CitLCYb transcripts. Even in green fruit, high levels of beta,epsilon-carotenoids and CitLCYe transcripts were not observed in the juice sacs. As fruit maturation progressed in Satsuma mandarin and Valencia orange, a simultaneous increase in the expression of genes (CitPSY, CitPDS, CitZDS, CitLCYb, CitHYb, and CitZEP) led to massive beta,beta-xanthophyll (beta-cryptoxanthin, zea, and violaxanthin) accumulation in both the flavedo and juice sacs. The gene expression of CitCRTISO was kept low or decreased in the flavedo during massive beta,beta-xanthophyll accumulation. In the flavedo of Lisbon lemon and Satsuma mandarin, massive accumulation of phytoene was observed with a decrease in the transcript level for CitPDS. Thus, the carotenoid accumulation during citrus fruit maturation was highly regulated by the coordination of the expression among carotenoid biosynthetic genes. In this paper, the mechanism leading to diversity in beta,beta-xanthophyll compositions between Satsuma mandarin and Valencia orange was also discussed on the basis of the substrate specificity of beta-ring hydroxylase and the balance of expression between upstream synthesis genes (CitPSY, CitPDS, CitZDS, and CitLCYb) and downstream synthesis genes (CitHYb and CitZEP).

Plants are challenged by a variety of biotic or abiotic stresses, which can affect their growth and development, productivity, and geographic distribution. In order to survive adverse environmental conditions, plants have evolved various adaptive strategies, among which is the accumulation of metabolites that play protective roles. A well-established example of the metabolites that are involved in stress responses, or stress tolerance, is the low-molecular-weight aliphatic polyamines, including putrescine, spermidine, and spermine. The critical role of polyamines in stress tolerance is suggested by several lines of evidence: firstly, the transcript levels of polyamine biosynthetic genes, as well as the activities of the corresponding enzymes, are induced by stresses; secondly, elevation of endogenous polyamine levels by exogenous supply of polyamines, or overexpression of polyamine biosynthetic genes, results in enhanced stress tolerance; and thirdly, a reduction of endogenous polyamines is accompanied by compromised stress tolerance. A number of studies have demonstrated that polyamines function in stress tolerance largely by modulating the homeostasis of reactive oxygen species (ROS) due to their direct, or indirect, roles in regulating antioxidant systems or suppressing ROS production. The transcriptional regulation of polyamine synthesis by transcription factors is also reviewed here. Meanwhile, future perspectives on polyamine research are also suggested.

The present review highlights some recent advances regarding the function of polyamines in the environmental stress tolerance of plants. When exposed to adverse environmental stresses, such as salt, drought, low temperature, and ozone, the complex dynamic kinetics of polyamine biosynthesis was observed. Polyamines titers altered in different manners dependent upon several factors, such as plant species, tolerance or sensitivity to stress, and duration of stress. The exogenous addition of polyamines to stress-treated cells or tissues could lead to injury alleviation and growth promotion in most cases, although the effects varied between polyamines and among plant species. Key genes responsible for polyamine biosynthesis have been cloned from a variety of plant species, whose expressions following stress have been investigated on a molecular basis. Overexpression of the genes caused the modification of polyamine biosynthesis in the transformants coupled with enhancement of stress tolerance. All of these results seem to indicate that polyamines are an important component in a plant's response to stress and that they play a significant role in counteracting stress.

Genome editing is a powerful technique for genome modification in molecular research and crop breeding, and has the great advantage of imparting novel desired traits to genetic resources. However, the genome editing of fruit tree plantlets remains to be established. In this study, we describe induction of a targeted gene mutation in the endogenous apple phytoene desaturase (PDS) gene using the CRISPR/Cas9 system. Four guide RNAs (gRNAs) were designed and stably transformed with Cas9 separately in apple. Clear and partial albino phenotypes were observed in 31.8% of regenerated plantlets for one gRNA, and bi-allelic mutations in apple PDS were confirmed by DNA sequencing. In addition, an 18-bp gRNA also induced a targeted mutation. These CRIPSR/Cas9 induced-mutations in the apple genome suggest activation of the NHEJ pathway, but with some involvement also of the HR pathway. Our results demonstrate that genome editing can be practically applied to modify the apple genome.

Twenty-four flavonoids have been determined in 66 Citrus species and near-citrus relatives, grown in the same field and year, by means of reversed phase high-performance liquid chromatography analysis. Statistical methods have been applied to find relations among the species. The F ratios of 21 flavonoids obtained by applying ANOVA analysis are significant, indicating that a classification of the species using these variables is reasonable to pursue. Principal component analysis revealed that the distributions of Citrus species belonging to different classes were largely in accordance with Tanaka's classification system.

The two FLOWERING LOCUS T (FT)-like genes of apple (Malus x domestica Borkh.), MdFT1 and MdFT2, have been isolated and characterized. MdFT1 and MdFT2 were mapped, respectively, on distinct linkage groups (LGs) with partial homoeology, LG 12 and LG 4. The expression pattern of MdFT1 and MdFT2 differed in that MdFT1 was expressed mainly in apical buds of fruit-bearing shoots in the adult phase, with little expression in the juvenile tissues, whereas MdFT2 was expressed mainly in reproductive organs, including flower buds and young fruit. On the other hand, both genes had the potential to induce early flowering since transgenic Arabidopsis, which ectopically expressed MdFT1 or MdFT2, flowered earlier than wild-type plants. Furthermore, overexpression of MdFT1 conferred precocious flowering in apple, with altered expression of other endogenous genes, such as MdMADS12. These results suggest that MdFT1 could function to promote flowering by altering the expression of those genes and that, at least, other genes may play an important role as well in the regulation of flowering in apple. The long juvenile period of fruit trees prevents early cropping and efficient breeding. Our findings will be useful information to unveil the molecular mechanism of flowering and to develop methods to shorten the juvenile period in various fruit trees, including apple.

Twenty-seven Citrus flavonoids were examined for their antiproliferative activities against several tumor and normal human cell lines. As a result, 7 flavonoids were judged to be active against the tumor cell lines, while they had weak antiproliferative activity against the normal human cell lines. The rank order of potency was luteolin, natsudaidain, quercetin, tangeretin, eriodictyol, nobiletin, and 3,3',4',5,6,7,8-heptamethoxyflavone. The structure-activity relationship established from comparison among these flavones and flavanones showed that the ortho-catechol moiety in ring B and a C2-C3 double bond were important for the antiproliferative activity. As to polymethoxylated flavones, C-3 hydroxyl and C-8 methoxyl groups were essential for high activity.

An SSR-based linkage map was constructed in Brassica rapa. It includes 113 SSR, 87 RFLP, and 62 RAPD markers. It consists of 10 linkage groups with a total distance of 1005.5 cM and an average distance of 3.7 cM. SSRs are distributed throughout the linkage groups at an average of 8.7 cM. Synteny between B. rapa and a model plant, Arabidopsis thaliana, was analyzed. A number of small genomic segments of A. thaliana were scattered throughout an entire B. rapa linkage map. This points out the complex genomic rearrangements during the course of evolution in Cruciferae. A 282.5-cM region in the B. rapa map was in synteny with A. thaliana. Of the three QTL (Crr1, Crr2, and Crr4) for clubroot resistance identified, synteny analysis revealed that two major QTL regions, Crr1 and Crr2, overlapped in a small region of Arabidopsis chromosome 4. This region belongs to one of the disease-resistance gene clusters (MRCs) in the A. thaliana genome. These results suggest that the resistance genes for clubroot originated from a member of the MRCs in a common ancestral genome and subsequently were distributed to the different regions they now inhabit in the process of evolution.

We determined the genome sequence of sweet cherry (Prunus avium) using next-generation sequencing technology. The total length of the assembled sequences was 272.4 Mb, consisting of 10,148 scaffold sequences with an N50 length of 219.6 kb. The sequences covered 77.8% of the 352.9 Mb sweet cherry genome, as estimated by k-mer analysis, and included >96.0% of the core eukaryotic genes. We predicted 43,349 complete and partial protein-encoding genes. A high-density consensus map with 2,382 loci was constructed using double-digest restriction site-associated DNA sequencing. Comparing the genetic maps of sweet cherry and peach revealed high synteny between the two genomes; thus the scaffolds were integrated into pseudomolecules using map- and synteny-based strategies. Whole-genome resequencing of six modern cultivars found 1,016,866 SNPs and 162,402 insertions/deletions, out of which 0.7% were deleterious. The sequence variants, as well as simple sequence repeats, can be used as DNA markers. The genomic information helps us to identify agronomically important genes and will accelerate genetic studies and breeding programs for sweet cherries. Further information on the genomic sequences and DNA markers is available in DBcherry (http://cherry.kazusa.or.jp (8 May 2017, date last accessed)).

BACKGROUND: Green tea has been shown to have beneficial effects against a variety of diseases such as cancer, obesity, diabetes, cardiovascular disease, and neurodegenerative diseases. Through cellular, animal, and human experiments, green tea and its major component, epigallocatechin-3-gallate (EGCG) have been demonstrated to have anti-inflammatory effects. Our previous findings have indicated that green tea and EGCG suppress the gene and/or protein expression of inflammatory cytokines and inflammation-related enzymes. METHODS: Using bibliographic databases, particularly PubMed (provided by the http://www.ncbi.nlm.nih.gov/pubmed, US National Library of Medicine, National Institutes of Health, United States), we examined the potential usefulness of green tea/EGCG for the prevention and treatment of inflammatory diseases in human clinical and epidemiological studies. We also reviewed results from cellular and animal experiments and proposed action mechanisms. RESULTS: Most of the results from the human studies indicated the beneficial effects of green tea and tea catechins against inflammatory diseases. The cellular and animal studies also provided evidence for the favorable effects of green tea/EGCG. These results are compatible with our previous findings and can be largely explained by a mechanism wherein green tea/EGCG acts as an antioxidant to scavenge reactive oxygen species, leading to attenuation of nuclear factor-κB activity. CONCLUSION: Since green tea and EGCG have multiple targets and act in a pleiotropic manner, we may consider their usage to improve the quality of life in patients with inflammatory disease. Green tea and EGCG have beneficial health effects and no severe adverse effects; however, care should be taken to avoid overdosage, which may induce deleterious effects including hepatic injury.

White root rot, caused by the ascomycete Rosellinia necatrix, is a devastating disease worldwide, particularly in fruit trees in Japan. Here we report on the biological and molecular properties of a novel bipartite double-stranded RNA (dsRNA) virus encompassing dsRNA-1 (8,931 bp) and dsRNA-2 (7,180 bp), which was isolated from a field strain of R. necatrix, W779. Besides the strictly conserved 5' (24 nt) and 3' (8 nt) terminal sequences, both segments show high levels of sequence similarity in the long 5' untranslated region of approximately 1.6 kbp. dsRNA-1 and -2 each possess two open reading frames (ORFs) named ORF1 to -4. Although the protein encoded by 3'-proximal ORF2 on dsRNA-1 shows sequence identities of 22 to 32% with RNA-dependent RNA polymerases from members of the families Totiviridae and Chrysoviridae, the remaining three virus-encoded proteins lack sequence similarities with any reported mycovirus proteins. Phylogenetic analysis showed that the W779 virus belongs to a separate clade distinct from those of other known mycoviruses. Purified virions approximately 50 nm in diameter consisted of dsRNA-1 and -2 and a single major capsid protein of 135 kDa, which was shown by peptide mass fingerprinting to be encoded by dsRNA-1 ORF1. We developed a transfection protocol using purified virions to show that the virus was responsible for reduction of virulence and mycelial growth in several host strains. These combined results indicate that the W779 virus is a novel bipartite dsRNA virus with potential for biological control (virocontrol), named Rosellinia necatrix megabirnavirus 1 (RnMBV1), that possibly belongs to a new virus family.

We investigated the mechanism of resistance to demethylation inhibitors (DMI) in Penicillium digitatum by isolating the CYP51 gene, which encodes the target enzyme (P450(14DM)) of DMI, from three DMI-resistant and three DMI-sensitive strains. The structural genes of all six strains were identical, but in the promoter region, a unique 126-bp sequence was tandemly repeated five times in the DMI-resistant strains and was present only once in the DMI-sensitive strains. Constitutive expression of CYP51 in the resistant strains was about 100-fold higher than that in the sensitive strains. We introduced CYP51, including the promoter region, from a DMI-resistant strain into a DMI-sensitive strain, which rendered the transformants DMI resistant and increased CYP51 expression. We also found that if the number of copies of the repeat was reduced to two, resistance and CYP51 expression also decreased. These results indicate that the 126-bp unit acts as a transcriptional enhancer and that a tandem repeat of the unit enhances CYP51 expression, resulting in DMI resistance. This is a new fungicide resistance mechanism for filamentous fungi.

Abstract We carried out a quantitative detection of Candidatus Liberibacter asiaticus, the bacterium associated with the disease of huanglongbing, in the vector psyllid Diaphorina citri by using a TaqMan real‐time PCR assay. The concentration of the bacterium was monitored at 5‐day intervals for a period of 20 days after psyllids were exposed as fifth instar nymphs or adults to a Ca . L. asiaticus‐infected plant for an acquisition access period of 24 h. When adults fed on Ca. L. asiaticus‐infected plant, the concentration of the bacterium did not increase significantly and the pathogen was not transmitted to any citrus seedlings. In contrast, when psyllids fed on infected plant as nymphs, the concentration of the pathogen significantly increased by 25‐, 360‐ and 130‐fold from the initial acquisition day to 10, 15 and 20 days, respectively. Additionally, the pathogen was successfully transmitted to 67% of citrus seedlings by emerging adults. Our data suggested that multiplication of the bacterium into the psyllids is essential for an efficient transmission and show that it is difficult for adults to transmit the pathogen unless they acquire it as nymphs.

Because fruit trees such as apple ( Malus × domestica Borkh.) flower and set fruit only after an extended juvenile phase lasting several years, efficient breeding of fruit trees is limited. We previously suggested that MdTFL1 ( Malus × domestica TFL1 ) functions analogously to TERMINAL FLOWER 1 ( TFL1 ) and that MdTFL1 is involved in the maintenance of the juvenile/vegetative phase in apple. To clarify the function of MdTFL1 in apple, we produced transgenic `Orin' apple trees expressing MdTFL1 antisense RNA. One of them flowered only 8 months after the transfer to the greenhouse, whereas the nontransformed control plants have not flowered in nearly 6 years. As expected, the expression of endogenous MdTFL1 was suppressed in the transgenic lines that showed precocious flowering. In addition, the expression level of the transgene was correlated with the reduction of the juvenile phase. These findings confirm that MdTFL1 functions like TFL1 and that MdTFL1 maintains the juvenile and vegetative phase in apple. Flower organs of the transgenic apple trees were normal in appearance, and a precocious flowering transgenic line set fruit and seeds. Interestingly, some flowers of the transgenic apple trees developed without undergoing dormancy. The expression of MdTFL1 in apple may affect flower development as well as flower induction.

To clarify the involvement of the arginine decarboxylase (ADC) pathway in the salt stress response, the polyamine titre, putrescine biosynthetic gene expression, and enzyme activities were investigated in apple [Malus sylvestris (L.) Mill. var. domestica (Borkh.) Mansf.] in vitro callus under salt stress, during recovery after stress, and when ADC was inhibited by D-arginine, an inhibitor of ADC. Salt stress (200 mM NaCl) caused an increase in thiobarbituric acid-reactive substances (TBARS) and electrolyte leakage (EL) of the callus, which was accompanied by an increase in free putrescine content, during 7 d of treatment. Conjugated putrescine was also increased, but this increase was limited to the early stage of salt stress. Accumulation of putrescine was in accordance with induction of ADC activity and expression of the apple ADC gene (MdADC). When callus that had been treated with 200 mM NaCl was transferred to fresh medium with (successive stress) or without (recovery) NaCl, TBARS and EL were significantly reduced in the recovery treatment, indicating promotion of formation of new callus cells, compared with the successive stress treatment. Meanwhile, MdADC expression and ADC activity were also decreased in the callus undergoing recovery, whereas those of the callus under successive stress were increased. Ornithine decarboxylase (ODC) activity showed a pattern opposite to that of ADC in these conditions. D-Arginine treatment led to more serious growth impairment than no treatment under salt stress. In addition, accumulation of putrescine, induction of MdADC, and activation of ADC in D-arginine-treated callus were not comparable with those of the untreated callus. Exogenous addition of putrescine could alleviate salt stress in terms of fresh weight increase and EL. All of these findings indicated that the ADC pathway was tightly involved in the salt stress response. Accumulation of putrescine under salt stress, the possible physiological role of putrescine in alleviating stress damage, and involvement of MdADC and ADC in response to salt stress are discussed.

Several studies have suggested that flavan-3-ols/procyanidins are associated with a reduced risk of developing obesity and metabolic syndrome. However, the role of highly polymeric procyanidins (PP), which are major non-absorbable flavonoids, in the biological effects, is not completely understood. Here, we show that 0.5% PP administration for 20 weeks alleviated obesity and regulate expression of genes related to lipid metabolism in C57BL/6J mice fed a high-fat/high-sucrose diet. PP-treatment attenuated weight gain and inflammatory effects including lipopolysaccharide and gut permeability. Additionally, metabolic urine profiling using high-performance liquid chromatography-quadrupole time-of-flight/mass spectrometry demonstrated that PP-treatment decreased the levels of endogenous metabolites associated with insulin resistance. Furthermore, microbial 16S rRNA gene sequencing of the cecum demonstrated that PP administration markedly decreased the Firmicutes/Bacteroidetes ratio and increased eight times the proportion of Akkermansia. These data suggest that PPs influence the gut microbiota and the intestinal metabolome to produce beneficial effects on metabolic homeostasis.

RNA-guided genome editing using the CRISPR/Cas9 CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) system has been applied successfully in several plant species. However, to date, there are few reports on the use of any of the current genome editing approaches in grape-an important fruit crop with a large market not only for table grapes but also for wine. Here, we report successful targeted mutagenesis in grape (Vitis vinifera L., cv. Neo Muscat) using the CRISPR/Cas9 system. When a Cas9 expression construct was transformed to embryonic calli along with a synthetic sgRNA expression construct targeting the Vitis vinifera phytoene desaturase (VvPDS) gene, regenerated plants with albino leaves were obtained. DNA sequencing confirmed that the VvPDS gene was mutated at the target site in regenerated grape plants. Interestingly, the ratio of mutated cells was higher in lower, older, leaves compared to that in newly appearing upper leaves. This result might suggest either that the proportion of targeted mutagenized cells is higher in older leaves due to the repeated induction of DNA double strand breaks (DSBs), or that the efficiency of precise DSBs repair in cells of old grape leaves is decreased.