Bud dormancy is a critical developmental process for perennial plant survival, and also an important physiological phase that affects the next season’s growth of temperate fruit trees. Bud dormancy is regulated by multiple genetic factors, and affected by various environmental factors, tree age and vigor. To understand the molecular mechanism of bud dormancy in Japanese apricot (Prunus mume Sieb. et Zucc.), we constructed a custom oligo DNA microarray covering the Japanese apricot dormant bud ESTs referring to the peach (P. persica) genome sequence. Because endodormancy release is a chilling temperature-dependent physiological event, genes showing chilling-mediated differential expression patterns are candidates to control endodormancy release. Using the microarray constructed in this study, we monitored gene expression changes of dormant vegetative buds of Japanese apricot during prolonged artificial chilling exposure. In addition, we analyzed seasonal gene expression changes. Among the 58539 different unigene probes, 2345 and 1059 genes were identified as being more than twofold up-regulated and down-regulated, respectively, following chilling exposure for 60 days (P < 0.05). Cluster analysis suggested that the expression of the genes showing expression changes by artificial chilling exposure were coordinately regulated by seasonal changes. The down-regulated genes included P. mume DORMANCY-ASSOCIATED MADS-box genes, which supported previous quantitative RT-PCR and EST analyses showing that these genes are repressed by prolonged chilling exposure. The genes encoding lipoxygenase were markedly up-regulated by prolonged chilling. Our parametric analysis of gene-set enrichment suggested that genes related to jasmonic acid (JA) and oxylipin biosynthesis and metabolic processes were significantly up-regulated by prolonged chilling, whereas genes related to circadian rhythm were significantly down-regulated. The results obtained from microarray analyses were verified by quantitative RT-PCR analysis of selected genes. Taken together, we have concluded that the microarray platform constructed in this study is applicable for deeper understanding of the molecular network related to agronomically important bud physiology, including dormancy release.

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Removing leaves around the grape cluster of Muscat Bailey A [Vitis × labruscana (Bailey) and Vitis vinifera (Muscat Hamburg)] cultivated in pergola style at the start of veraison increased the photosynthetically active radiation value by more than approximately 60- and 30-fold compared with those of the control and grape cluster from vines grown on ground covered with reflective film (reflective-film-treated grape cluster), respectively. The improved light exposure caused by leaf removal increased total anthocyanin concentration and changed the ratios of anthocyanin derivatives in the grape skins. Total anthocyanin concentration in the leaf-removal-treated grape skins 10 weeks after veraison increased by approximately 6.5-fold compared with that of the control. In addition, delphinidin-based anthocyanin concentrations in the leaf-removal-treated grape skins increased by approximately 7-fold compared with those of the control. Leaf removal up-regulated anthocyanin-synthesis-related genes in grape skins, such as CHS, F3′H, F3′,5′H, and UFGT. In particular, the overexpression of F3′,5′H in the leaf-removal-treated grape skins suggested that leaf removal contributed to the accumulation of delphinidin-based anthocyanins in grape skin. These findings are expected to improve viticultural practices with the aim of producing dark-colored red wine made from cultivars grown in pergola style.

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Apple latent spherical virus (ALSV) vectors have been shown to effectively induce stable virus-induced gene silencing (VIGS) in a wide range of plant species, including rosaceous fruit tree species such as apple (Malus × domestica Borkh.), pear (Pyrus communis L.), and Japanese pear (P. pyrifolia Nakai). In this study, we attempted to develop a VIGS-based gene evaluation system for two Prunus fruit tree species, apricot and Japanese apricot, using ALSV vectors. A partial sequence of the P. armeniaca PHYTOENE DESATURASE (ParPDS) gene was cloned and ligated into the T-DNA region of a binary vector, pBICAL2, designed based on RNA2 of ALSV. The resultant pBICAL2-ParPDS was introduced into a disarmed Agrobacterium strain, EHA105. pBICAL1, a binary plasmid for the expression of ALSV RNA1 in plants, was also introduced into EHA105. Leaves of Nicotiana benthamiana were infected with pBICAL1/EHA105 and pBICAL2-ParPDS/EHA105 simultaneously to produce and amplify recombinant ALSV particles. The amplified ParPDS-ALSV in N. benthamiana was isolated and infected into the cotyledons of apricot and Japanese apricot seedlings by particle bombardment. Although our attempts to infect wild and recombinant ALSVs into Japanese apricot seedlings were unsuccessful, uniform discoloration of the upper leaves, a typical phenotype of PDS knock down, was observed several weeks after inoculation in apricot seedlings. We discuss the possible use of this VIGS-based gene evaluation system in Prunus.

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The expression profiles of carotenoid metabolism pathway genes are one of the important factors that regulate carotenoid content and composition in the fruit of Citrus cultivars. To identify regulatory factors of gene expressions in the carotenoid pathway, expression quantitative trait loci (eQTL) analysis was applied using a hybrid population. In the analyzed population, significant eQTLs for phytoene synthase (PSY), phytoene desaturase (PDS), ζ-carotene desaturase (ZDS), β-ring hydroxylase (HYb), and zeaxanthin epoxidase (ZEP) were detected. Among these eQTLs, eQTLs for HYb and ZEP were located on their responsible gene loci, respectively. This result indicates that the expression level of HYb and ZEP was influenced by cis-elements in their up-stream regions. Conversely, eQTLs for PDS and ZDS were located in different loci from those of the responsible genes, indicating that the loci were trans-regulating factors. It also suggested that expression levels of PDS and ZDS were regulated by a common transcription factor because their eQTLs were co-localized. Significant eQTL of lycopene β-cyclase (LCYb) and 9-cis-epoxycarotenoid dioxygenase (NCED) were not detected in this population. The major factors to control gene expression depend on each carotenoid metabolism gene, and their combination might cause complex carotenoid metabolism and extend the variation of content and composition in citrus varieties. eQTL analysis was demonstrated as a powerful tool to identify cis- or trans-regulation for carotenoid metabolism genes, as well as in generating functionally defined markers for marker-assisted selection to increase the carotenoid content of citrus in breeding programs.

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The absorption of NO₃⁻ and growth of radish (Raphanus sativus L. ‘Yukikomachi’), and the timing and interval of NO₃⁻ supply were examined to evaluate quantitative nutrient management (QNM) of nutrient solution in a hydroponic culture of radish plants. In experiment 1, the amount of NO₃⁻ required for growth to a marketable size (30–35 g FW of thickened axis) was presumed to be approximately 1000 mg/plant by the direct measurement of NO₃⁻ absorption of radish plants grown with the EC-based control management method (EC-based control method) of nutrient solution containing different concentrations (2, 4, 6, 8, and 10 me·L⁻¹) of NO₃⁻. In Experiment 2, plants were supplied with the total amount of NO₃⁻ (1000 mg/plant) at the beginning of the experiment or with 1/5 of the total amount of NO₃⁻ (1000 mg/plant) repeatedly 5 times every 4 days, and then their fresh weight and nutrient absorption were compared with the plants grown with the EC-based control method. Significant differences in the growth of thickened axes and leaves were not obtained among plants grown by three different methods. However, plants appeared to be supplied with an excess amount of nutrients because EC and NO₃⁻ levels were high at the end of cultivation. From the experiment in which plants were supplied with the whole amount of mineral nutrients containing 900, 800, and 700 mg/plant of NO₃⁻ at the beginning of the experiment in December, it became apparent that 800 mg/plant of nitrate would be sufficient for radish growth in the cold season. In conclusion, we propose the QNM method supplying the whole amount of nutrients required for crop growth at the beginning of cultivation so that radish plants could be produced without draining nutrient solution containing a large amount of NO₃⁻ from the hydroponic system into the environment.

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Japanese old azalea cultivars have various floral mutations. We isolated MADS-box class C homologous genes from wild-type Rhododendron macrosepalum to analyze the expression patterns in the floral organs of the narrow-petal mutational cultivars ‘Hanaguruma’, ‘Gin-no-zai’, and ‘Seigaiha’. The AG homologous genes RmAG1-1/-2/-3 were 99–100% identical to RkAG1-1/-2, which was isolated from R. kaempferi. In ‘Hanaguruma’, ‘Seigaiha’, and the wild type, the relative expression level of RmAG in whorl 2 was much lower than that in whorl 3. In contrast, the relative expression level of RmAG in whorl 2 of ‘Gin-no-zai’ was approximately 15.5% of that in whorl 3. In ‘Gin-no-zai’, the petals with mutations were categorized as 1 of these 3 types: type 1, consisting of narrow petals with traces of anthers; type 2, consisting of narrow petals only; and type 3, consisting of petals that coalesced with the neighboring petals. The relative expression levels of RmAG gradually increased from type 3 to type 1 petals. These results suggest that the degree of staminoidy for the petals in whorl 2 is attributable to the expression levels of RmAG.

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Cut Eustoma grandiflorum (Raf.) Shinn. flowers are produced year-round in Japan; however, winter conditions are not favorable for flower production due to low sunlight levels. Here, we investigated the effect of CO₂ enrichment after the flower budding stage on the growth and flowering of Eustoma ‘Bolero White’, which were grown under forcing culture for winter shipping. CO₂ enrichment increased the fresh weight of plants, in addition to increasing the dry weights of leaves, stems, flower buds and open flowers, and roots. CO₂ enrichment also increased the relative growth rate (RGR) by 32%, due to the net assimilation rate (NAR) being stimulated. However, CO₂ enrichment had no effect on plant height or the leaf area ratio (LAR). Furthermore, CO₂ enrichment increased the total number of flower buds and open flowers, in addition to accelerating flower bud development and the promotion of flowering. During the period of enrichment, the vegetative organs continued to grow in CO₂-enriched plants, but not in the control plants. In conclusion, CO₂ enrichment promoted flowering and improved the quality of cut flowers (i.e., increasing plant fresh and dry weight and the total number of flower buds and open flowers) of Eustoma under low-sunlight winter conditions.

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Three new acylated cyanidin 3,7-diglucosides (1–3) were isolated from the red-purple flowers of Sophronitis wittigiana as its main floral anthocyanins. These three pigments were based on cyanidin 3,7-diglucoside as the deacylanthocyanin, and their structures were determined to be cyanidin 3-O-[6-O-(malonyl)-β-glucopyranoside)]-7-O-[6-O-(trans-caffeoyl)-β-glucopyranoside] as pigment 1, its demalonyl anthocyanin as pigment 2, and cyanidin 3-O-[6-O-(malonyl)-glucoside]-7-O-glucoside as pigment 3 by chemical and spectroscopic methods. On the other hand, five known acylated cyanidin 3,7,3′-triglucosides (4–8) were isolated from orange-red or red flowers of S. acuensis, S. brevipedunculata, S. cernua, S. coccinea var. xanthoglossa, and S. grandiflora as well as those of S. coccinea, and identified to be pigment 4 as Sophronitis coccinea anthocyanin 1 (SCA 1), pigment 5 as SCA 2, pigment 6 as SCA 3, pigment 7 as SCA 4, and pigment 8 as SCA 5 in comparison of the TLC, HPLC, and UVVis properties with standard samples of SCAs 1–5. These results showed that the three 3,7,3′-O-hydroxy groups of anthocyanins were all substituted with acylglucose and/or glucose residues in the orange-red or red flowers of six taxa of Sophronitis, whereas the 3′-O-hydroxy group was free from glucose in the red-purple flowers of S. wittigiana. Thus, the inactivation of 3′-O-glycosylation in cyanidin units might be involved in causing the blue color direction shift from orange-red or red to red-purple flower color of S. wittigiana.

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2,4-Pyridinedicarboxylic acid (PDCA) is a structural analog of 2-oxoglutarate and has been shown to inhibit 2-oxoglutarate-dependent dioxygenases by competing with 2-oxoglutarate, and ethylene production in detached carnation flowers by competing with ascorbate on 1-aminocyclopropane-1-carboxylate (ACC) oxidase action. In the present study, the inhibition of ACC oxidase action by PDCA was confirmed with a recombinant enzyme produced in Escherichia coli from carnation DcACO1 cDNA. PDCA had various effects on ethylene production in cut ‘Light Pink Barbara (LPB)’ carnation flowers; ethylene production was accelerated or delayed in some flowers, whereas it did not change in others as compared to untreated control flowers. This varied action of PDCA may be caused by its possible combined actions; that is, inhibition of ACC oxidase action as well as its action on unidentified biochemical processes which use 2-oxoglutarate as a co-substrate, such as the biosynthesis and inactivation of gibberellins. Meanwhile, PDCA treatment significantly prolonged the vase life of bunches of cut ‘LPB’ carnation flowers; the magnitude of the extension of vase life was 53, 111, and 135% at 0.3, 1, and 2 mM PDCA, respectively, as compared with the non-treated control. Also, PDCA lengthened the vase life of ‘Mule’ carnation flowers. The present findings suggest the potential of PDCA as a preservative for cut flowers of spray carnations.

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Japanese apricot (Prunus mume Siebold & zucc.) fruits of ‘Nanko’ have softer flesh than those of ‘Gojiro’. Therefore, there are differences in the processability of these cultivars. We investigated the characteristics of cell-wall polysaccharide degradation that significantly affects fruit firmness in these 2 cultivars at unripe, ripe, and drop stages. Fruit firmness of ‘Gojiro’ was maintained during unripe and ripe stages and decreased after the ripe stage, while that of ‘Nanko’ decreased constantly and was lower than that of ‘Gojiro’ at ripe and drop stages. Amount of the pectin and the hemicellulose substances decreased commonly in ‘Gojiro’ and ‘Nanko’ in the process of fruit softening, in addition, decrease in mol wt of these polysaccharides significantly synchronized with decrease in fruit firmness, suggesting that degradation of the pectin and the hemicellulose cause the softening of Japanese apricot fruits. The amount of neutral sugar (NS) and uronic acid (UA) in the pectin fraction and NS in the hemicellulose fraction in ‘Nanko’ decreased more than in ‘Gojiro’. Moreover, the cellulose content of ‘Nanko’ decreased during the fruit-softening process. These results suggest that higher degradation of pectin, hemicellulose, and cellulose in ‘Nanko’ fruits would result in softer fruits than ‘Gojiro’.

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