Megumi Ishimaru

Myb-related genes of the Kyoho grape (Vitis labruscana) regulate anthocyanin biosynthesis

Abstract. Partial cDNAs of myb-related regulatory genes were isolated from the tetraploid Kyoho grape (Vitis labruscana: V. labrusca × 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.

Comparison of UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT) gene sequences between white grapes (Vitis vinifera) and their sports with red skin.

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.

Abscisic acid and 2,4-dichlorophenoxyacetic acid affect the expression of anthocyanin biosynthetic pathway genes in ‘Kyoho’ grape berries

Summary The 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.

Expression of a xyloglucan endo-transglycosylase gene is closely related to grape berry softening

Abstract 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.

Regulation of fermentative metabolism in tomato fruit under low oxygen stress

Summary Tomato fruit was stored under a continuous flow of 0% and 3% O2 (balance N2) or air for 7 d at 20°C to study the regulation of fermentative metabolism. The concentrations of ethanol and acetaldehyde were very low during storage at 3% O2 and air. At 0% O2, ethanol and to a lesser extent acetaldehyde, rapidly accumulated in the tissue. However, lactate concentration did not change during the experiment and was not significantly influenced by O2 concentration. Pyruvate decarboxylase (PDC) activity greatly increased in fruit exposed to 3% O2, while activity in fruit exposed to 0% O2 was the same level as the control. Alcohol dehydrogenase (ADH) activity greatly increased in fruit exposed to 3%, but at 0% O2 was the same level as the control. Lactate dehydrogenase (LDH) activity greatly increased in fruit exposed to 3%, but at 0% O2 was the same level as the control. The ADH activity in tomato fruit was about ten times greater than that of PDC activity and about 100 times greater than that of LDH activity during storage. Concentration of NADH in fruit exposed to 3% O2 was greater than that in fruit exposed to 0% O2 and air. Concentrations of pyruvate did not change during storage at 1% O2 and air. At 0% O2, pyruvate rapidly accumulated in its tissue. The K m of ADH in tomato fruit was 0.28 mM for acetaldehyde, and 0.058 mM for NADH. The Km of PDC in tomato fruit was 0.38 mM for pyruvate. The Km of LDH in tomato fruit was 0.18 mM for pyruvate. Possible regulation of fermentative metabolism is briefly considered.

Physiological responses and quality attributes of Japanese pear 'Kosui' fruit kept in low oxygen atmospheres

Summary Japanese pear ‘Kosui’ fruits were stored under a continuous flow of 0%, 1%, 3%, 5% and 10% O2 (balance N2) or air for 7 d at 20°C to study the effects of low O2 on their physiological responses and quality attributes. Low O2 treatments did not significantly influence changes in skin colour and soluble solids content. However, weak off-flavours were detected in the fruits stored at 0% O2 on day 3, and the intensity of these off-flavours increased as storage progressed. The concentrations of acetaldehyde in fruit increased throughout the storage period. The ethanol concentration was greatly increased in fruits stored at 0% O2. Moreover, ethanol concentrations were much higher than those of acetaldehyde and remained very low during storage in air, but their concentration were just slightly increased in fruits exposed to 1%, 3%, 5% and 10% O2. Pyruvate decarboxylase activity was greatly increased in fruits exposed to 1% and 3% O2, while its activity in fruits exposed to 5% and 10% O2 were only slightly higher than that of the control and at 0% O2 at the same level as the control. Alcohol dehydrogenase (ADH) activity greatly increased in fruit exposed to 0%, 1%, 3% and 5% O2, while at 10% O2, ADH was only slightly higher than the control. Changes in ADH isozymes correlated well with changes in ADH activity. The homogenate pH of fruits exposed to 1%, 3%, 5% and 10% O2 and air remained constant, while in fruit stored at 0% O2 their pH increased. The potential for using low O2 atmospheres to help in maintaining the quality of Japanese pear ‘Kosui’ is discussed.