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Tomomi Tsuda, Masami Yamaguchi, Chikako Honda, and Takaya Moriguchi

We used RNA blot analysis to examine the expression of six genes of the anthocyanin biosynthesis pathway in the flowers and fruit skins at three developmental stages of white and red peaches and a deep-red nectarine [Prunus persica (L.) Batch]. In the red peach `Akatsuki' and the deep-red nectarine `Flavortop', expression levels of anthocyanin biosynthesis genes were related to anthocyanin accumulation in the fruit skin; expression of all six genes dramatically increased at Stage III of fruit development, and anthocyanin concentration also increased at this stage. In the white peach `Mochizuki', however, expression of the chalcone synthase gene (CHS) and the dihydroflavonol 4-reductase gene (DFR) was undetectable in Stage III, although the chalcone isomerase gene (CHI), the flavanone 3-hydroxylase gene (F3H), the anthocyanidin synthase gene (ANS), and the UDP-glucose-flavonoid 3-O-glucosyltransferase gene (UFGT) were expressed. We occasionally found red pigment in the skin of `Mochizuki' peach. In these red skin areas, both CHS and DFR were clearly expressed in Stage III. These results suggest that CHS and DFR are the key regulatory genes in the process of anthocyanin biosynthesis in mature red peach and nectarine.

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Satoru Kondo, Kentaro Hiraoka, Shozo Kobayashi, Chikako Honda, and Norihiko Terahara

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.

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Hiroyasu Kitashiba*, Yu-Jin Hao, Chikako Honda, Masayuki Kita, and Takaya Moriguchi

Polyamines [putrescine (put), spermidine (spd), and spermine (spm)] are aliphatic amines that are implicated in the regulation of many basic physiological processes such as cell growth, proliferation and stress responses in organisms including plants (Walden et al. 1997). Put is metabolized to spd and spm through the successive enzymatic reactions of spd synthase (SPDS) and spm synthase (SPMS) with the use of decarboxylated S-adenosylmethionine (dcSAM) as an aminopropyl donor, which is generated by SAM decarboxylase (SAMDC). So far, two MdSAMDC (MdSAMDC1 and MdSAMDC2) homologous to SAMDC and two MdACL5 (MdACL5-1 and MdACL5-2) homologous to ACL5 encoding SPMS in Arabidopsis (Hanzawa et al. 2000) were isolated from `Orin' apple. To investigate the function of these genes, complementation analyses were carried out using yeast mutants. Each of the MdSAMDCs consists of three ORFs; tiny- and small-ORFs in the 5' regions, and main ORF like other plant SAMDC genes. Both constructs for MdSAMDC containing all ORFs (SAM-DCall) or containing only main ORF (SAMDCorf) were capable of recovering the growth of yeast SAMDC-deficient mutants (delta spe2) without supplement of spd, although the SAMDCall constructs always showed the lower growth speed than the SAMDCorf constructs. On the other hand, yeast SPMS-deficient mutant (delta spe4) introduced by MdACL5 cDNA produced significantly higher amount of spm than the delta spe4 with control vector by HPLC. Collectively, these results suggest that both MdSAMDCs are functional as a SAMDC and the tiny- and small-ORFs are negative-regulatory factor for the translation efficiency of SAMDC, and also that MdACL5 encodes a functional SPMS like as ACL5 in Arabidopsis. The first and second authors contributed equally to this work.

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Chikako Honda, Hideo Bessho, Mari Murai, Hiroshi Iwanami, Shigeki Moriya, Kazuyuki Abe, Masato Wada, Yuki Moriya-Tanaka, Hiroko Hayama, and Miho Tatsuki

The objective of this study was to investigate the effects of temperature treatments on anthocyanin accumulation and ethylene production in the fruit of early- and medium-maturing cultivars that were harvested early during fruit ripening. We first investigated the effects of various temperature treatments on anthocyanin accumulation in detached apples of ‘Tsugaru’, ‘Tsugaru Hime’, ‘Akane’ and ‘Akibae’ using an incubator. Three years of experiments demonstrated that at harvest, the lower-temperature treatments induced anthocyanin accumulation in ‘Tsugaru’, ‘Tsugaru Hime’, and ‘Akibae’ fruits, whereas the increases in anthocyanin accumulation under the 25 °C treatment were similar to those under the 15 and 20 °C treatments in ‘Akane’ fruit. The rate of ethylene production did not increase substantially during the temperature treatments in any of the four cultivars, except after the treatments of ‘Tsugaru’ fruit at harvest. The inhibition of ethylene action by the application of 1-methylcyclopropene (1-MCP) to detached fruits at harvest suppressed anthocyanin development under 15 and 20 °C temperature treatments in ‘Tsugaru’, ‘Tsugaru Hime’, and ‘Akibae’, but not in ‘Akane’. In the second experiment, we investigated changes in the anthocyanin concentration in attached fruit of ‘Misuzu Tsugaru’ under different temperature conditions in a greenhouse. At harvest, the anthocyanin concentration in fruit under the hotter climatic condition (29 °C 12 hours/19 °C 12 hours) was lower than that under the control condition (25 °C 12 hours/15 °C 12 hours). During the last week before harvest, anthocyanin development proceeded rapidly in fruit skin not only under the control condition, but also under the hotter climatic condition. The rapid accumulation of anthocyanin in the fruit skin of ‘Misuzu Tsugaru’ at harvest under a relatively high temperature (25 °C) condition was confirmed by the experiment using an incubator. At harvest, the maximum level of ethylene production in fruits sampled from trees grown under the hotter climatic condition was 9-fold higher than that in fruits from trees grown under the control condition. These results indicate that the comparison of pigmentation potential after the 15 or 25 °C treatments using detached fruit was effective for estimating anthocyanin accumulation in fruit skins under hotter climatic conditions in early- and medium-maturing cultivars that were harvested early and that a hotter climatic condition during ripening increased ethylene production in apple fruit after harvest.