Masayuki Nozue

Selection of a High Anthocyanin-Producing Cell Line of Sweet Potato Cell Cultures and Identification of Pigments

Summary A high anthocyanin-producing cell line was selected from the callus cultures initiated with the root tissues of sweet potato ( Ipomoea batatas Lam. cv. Kintoki) with the naked eye. The selection of the cell lines was carried out using PRL-4C agar medium supplemented with 0.5 mg 1 −1 2,4-dichlorophenoxyacetic acid and 3 % sucrose under continuous illumination. Thirty-two selections brought about a 40-fold increase in anthocyanin content compared to the original callus. The high stability in anthocyanin productivity of the obtained cell line was maintained even after more than 35 non-selective subcultures. On the other hand, a pigment non-producing cell line was also obtained by the repeated selections of the non-pigmented cell clusters. The major anthocyanins produced in the anthocyanin-producing cell line were identified as acylated 3-sophoroside-5-glucoside of cyanidin and peonidin with caffeic, ferulic and phydroxybenzoic acid.

Race 3, a new race of Fusarium oxysporum f. sp. lactucae determined by a differential system with commercial cultivars

Abstract Pathogenic variation among 26 Japanese isolates of Fusarium oxysporum f. sp. lactucae (FOL) was tested using 21 lettuce cultivars to select commercial lettuce cultivars as race differential indicators. Cultivar Costa Rica No. 4 was resistant to race 1 but susceptible to race 2, consistent with the conventional standard differential line VP1010. Cultivar Banchu Red Fire was susceptible to race 1 but resistant to race 2, which showed an opposite type of reaction as another differential line VP1013. Cultivar Patriot was susceptible to both races. The resistance reactions of the three cultivars under field conditions were identical with that observed in the seedlings. Thus cv. Costa Rica No. 4 and cv. Banchu Red Fire can be used as differential hosts to identify pathogenic races of FOL. This differential system showed that all FOL isolates obtained from diseased butterhead lettuce in Fukuoka, Japan were new races (i.e., pathogenic to three cultivars). We propose that the new race be designated race 3. Isolates of FOL, the pathogen of Fusarium wilt in lettuce, obtained from California showed the same reaction as that of race 1. Furthermore, the Japanese isolate SB1-1 (race 1) and California isolate HL-2 belonged to the same vegetative compatibility group. Our results suggest that both of the fungi are the same forma specialis.

Expression of a Vacuolar Protein (VP24) in Anthocyanin- Producing Cells of Sweet Potato in Suspension Culture'

VP24, an abundant protein of 24 kD, was found to accumulate in the anthocyanin-containing vacuoles of cells of sweet potato (lpomoea batatas) in suspension culture. Light-induced expression of VP24 was analyzed by immunoblotting in three different cell lines that produced anthocyanins at different rates. The expression of VP24 was closely correlated with the accumulation of anthocyanin in these cell lines. Immunocytochemical detection of VP24 with specific antibodies on thin sections showed that VP24 was localized in the intravacuolar pigmented globules (cyanoplasts) in the anthocyanin-containing vacuoles and not in the tonoplast. No VP24 immunogold labeling was detected in the vacuoles of the cell line that does not produce anthocyanin. We suggest that VP24 may be involved in the formation of the cyanoplast via an interaction with anthocyanin, and that it may play an important role in the trapping in vacuoles of large amounts of anthocyanins that have been transported into these vacuoles.

In Planta Transformation of Kenaf Plants (Hibiscus cannabinus var. aokawa No. 3) by Agrobacterium tumefaciens

Kenaf was transformed by inoculation of Agrobacterium tumefaciens onto the meristems of young plants in pots. The transformation was demonstrated by three lines of evidence: a phenotypic inheritance from T(0) to T(1) plants, detection of the transgene in both T(0) and T(1) plants, and rescue of plasmids composed of T-DNA of the binary vector and flanking plant genomic DNA from T(1) plants.

Phylogenetic relationships between the lettuce root rot pathogen Fusarium oxysporum f. sp. lactucae races 1,2, and 3 based on the sequence of the intergenic spacer region of its ribosomal DNA

The genetic relationship between the vegetative compatibility groups (VCGs) and between physiological races of Fusarium oxysporum f. sp. lactucae (FOL), the causal pathogen of lettuce root rot, was determined by analyzing the intergenic spacer (IGS) region of its ribosomal DNA. A total of 29 isolates containing a type strain were tested: 24 Japanese isolates, 2 Californian isolates, and 3 Italian isolates. Three races (races 1, 2, and 3) were found in Japan, and race 1 was also distributed in California and Italy. Races 1, 2, and 3 each belonged to a distinct VCG: VCG-1, VCG-2, and VCG-3 (VCG-3-1, VCG-3-3), respectively. Phylogenetic (neighbor-joining) analysis of the IGS sequences revealed that races 1, 2, and 3 coincided with three phylogenetic groups (PG): PG-1, PG-2, and PG-3, respectively. These results indicate that the three races are genetically quite different and have a strong correlation with VCGs and phylogenetic groupings.

Occurrence of anthocyanoplasts in cell suspension cultures of sweet potato.

Intensely pigmented and spherical vesicles (anthocyanoplasts) were found in anthocyanin-containing cells of sweet potato (Ipomoea batatas) suspension cultures. Anthocyanin synthesis began to first occur 24–48 h after exposure to light, and then numerous small red vesicles were detected under a microscope. The frequency of anthocyanoplast-containing cells rapidly increased to finally about 80% of the total cultured cells after 5 days of irradiation. Fully developed anthocyanoplasts reached 10–15 μm in diameter. On the other hand, neither anthocyanin synthesis nor development of anthocyanoplasts was induced in the dark-cultured cells. 2,4-D also inhibited anthocyanin synthesis and development of these vesicles. The results suggest that anthocyanoplasts might be a site of anthocyanin synthesis and/or accumulation.

Expression Pattern and Gene Structure of Phenylalanine Ammonia-Lyase in Pharbitis nil

PAL gene expression were examined in flower buds and irradiated hypocotyls in Pharbitis nil. PAL activity and transcript levels were correlated with the accumulation of anthocyanin. Both in flower buds and hypocotyls, transcript levels, PAL activity, and then the amount of anthocyanin, increased. The PAL transcript was abundant in flower buds for a few days before flower opening. But the increase in PAL transcript induced by irradiation was temporal in hypocotyls. Phytochrome was shown to be involved in inducing the accumulation of anthocyanin in hypocotyls. To examine the mechanism regulating the expression of the PAL gene, the gene was cloned and sequenced, and the promoter region was compared with that of other PALs. The gene had two exons separated by an intron of 989 bp with consensus sequences at the intron/exon border. The predicted primary structure of the PAL protein consists of 711 amino acids. The promoter region was AT-rich and there were sequences similar to box 1, box 2, an AT-1 binding site and a G box. The role of PAL in the accumulation of anthocyanin is discussed.

Isolation and culture of protoplasts from high anthocyanin-producing callus of sweet potato

Optimum conditions for the isolation and culture of protoplasts from high anthocyanin-producing callus of sweet potato (Ipomoea batatas) were investigated. Growth phase of callus and the ratio of callus-enzyme solution affected the yield of protoplasts. Composition of the medium and protoplast density were examined for protoplast culture.Small colonies were regenerated from the protoplasts. Upon transfer to light a high amount of anthocyanin was accumulated in these colonies.

Effects of Temperature on the Pattern of Anthocyanin Accumulation in Seedlings of Polygonum cuspidatum

Polygonum cuspidatum seedling. Anthocyanin accumulated first in the lower part of hypocotyls and then the site of accumulation gradually extended toward the upper part of hypocotyls when seedlings were irradiated with white light (WL) at 25 C. Etiolated seedlings accumulated anthocyanin only in the upper parts (hook and cotyledons) when the seedlings were irradiated with WL at 5 C. De-etiolated seedlings that had been pre-irradiated with WL for 1 day at 25 C accumulated anthocyanin both in upper and lower parts of the seedlings when the seedlings were irradiated with WL at 5 C. Spectral sensitivity was dependent on the temperature during irradiation. Red light (R), blue light (B), and near ultra-violet light (NUV) induced the accumulation of anthocyanin at 5 C but only NUV was effective in inducing the accumulation of anthocyanin at 25 C. Dichlorophenyl dimethylurea (DCMU) inhibited WL-induced anthocyanin accumulation but did not NUV-induced anthocyanin accumulation at 25 C. However, sucrose promoted NUV action at 25 C, indicating that photosynthesis can promote NUV-induced anthocyanin accumulation. Distribution of phytochrome in etiolated seedlings, that was examined by spectrophotometry, was similar to the distribution of anthocyanin at 5 C. Furthermore, phytochrome remained after 48 hr irradiation with WL at 5 C although phytochrome was rapidly degraded at 25 C.

Detection and characterization of a 36-kDa peptide in C-terminal region of a 24-kDa vacuolar protein (VP24) precursor in anthocyanin-producing sweet potato cells in suspension culture.

A 24-kDa vacuolar protein (VP24) was found to accumulate in anthocyanin-producing sweet potato cells (Ipomoea batatas Lam.) in suspension culture [Nozue et al., Plant Physiol. 115 (1997) 1065]. VP24 cDNA (accession No. AB025531) encodes a 96.3-kDa large precursor protein with a C-terminal propeptide which contains the eight putative transmembrane domains. The mature VP24 is probably involved in the formation of intravacuolar pigmented globules (cyanoplasts) in highly anthocyanin-containing vacuoles, but the biological function of the C-terminal region including the putative transmembrane domains is unknown. To confirm the expression and characterize the C-terminal region in the VP24 protein precursor in the anthocyanin-producing cells, polyclonal antibodies were developed against the fusion protein, including the C-terminal region, expressed in Escherichia coli. Western blot analysis showed that a 36-kDa peptide (VP36) localized in anthocyanin-containing vacuoles was expressed under continuous illumination, but not in darkness. The expression pattern of VP36 showed high similarity to VP24. These results suggested that VP36 may be derived from the large VP24 protein precursor; it includes several of the hydrophobic domains in the C-terminal region.